Tissue factor and vascular endothelial growth factor expression in colorectal cancer: relation with cancer recurrence.

OBJECTIVE: This study was undertaken to quantify tissue factor (TF) and vascular endothelial growth factor (VEGF) in colorectal cancer and to evaluate their possible relationship with recurrence. METHOD: TF and VEGF were measured by enzyme-linked immunosorbent assay in surgical tumour specimens and normal mucosa from 50 consecutive patients with colorectal cancer who were followed up for 3 years for the assessment of disease recurrence. RESULTS: TF and VEGF antigens were detected in all tumour samples. VEGF, but not TF, was much higher in tumour than in normal mucosa (P < 0.0001), as also confirmed by measurement of specific mRNAs. There was a strong correlation between TF and VEGF antigens (P < 0.0005) in tumour tissue but not in normal mucosa. Neither protein was related to tumour stage, grade or size. Local or distant recurrence was statistically related to pTNM stage. High VEGF, but not TF, levels in tumour extracts were associated with an increased risk of recurrence both by univariate (RR, 4.00, 95% CI: 1.45-11.0) and multivariate analyses (RR, 3.65, 95% CI: 1.33-10.0). CONCLUSION: These findings suggest that VEGF content in colorectal cancer is an independent risk factor for tumour recurrence and might help select patients who might benefit from adjuvant therapy.

A novel nitric oxide-releasing statin derivative exerts an antiplatelet/antithrombotic activity and inhibits tissue factor expression.

BACKGROUND: NO-releasing statins are new chemical entities, combining HMG-CoA reductase inhibition and slow NO release, that possess stronger anti-inflammatory and antiproliferative activities than the native statins. OBJECTIVE: We evaluated the antithrombotic effects of nitropravastatin (NCX-6550) by assessing its activity on platelet activation and tissue factor (TF) expression by mononuclear cells in vitro and in vivo. METHODS AND RESULTS: In vitro, NCX-6550 inhibited (1) U46619- and collagen-induced platelet aggregation in buffer and plasma; (2) collagen-induced P-selectin expression in whole blood and (3) platelet adhesion to collagen-coated coverslips under high shear stress. These effects were displayed at concentrations of NCX-6550 ranging from 25 to 100 mum, and were totally reverted by the guanylylcyclase inhibitor ODQ (10 microm). Equimolar concentrations of pravastatin had no influence on these parameters of platelet function. LPS- and PMA-induced TF expression by blood mononuclear cells was also inhibited by NCX-6550 (IC50 13 microm), but not by pravastatin, as assessed by functional and immunological assays and by real-time PCR. In a mouse model of platelet pulmonary thromboembolism, induced by the i.v. injection of collagen plus epinephrine, pretreatment with NCX-6550 (24-48 mg kg(-1)) significantly reduced platelet consumption, lung vessel occlusion and mortality. Moreover, nitropravastatin markedly inhibited the generation of procoagulant activity by spleen mononuclear cells and peritoneal macrophages in mice treated with LPS. In these in vivo models too, pravastatin failed to affect platelet activation and monocyte/macrophage procoagulant activity. CONCLUSIONS: Our results show that nitropravastatin exerts strong antithrombotic effects in vitro and in vivo, and may represent an interesting antiatherothrombotic agent for testing in acute coronary syndromes.

Changes in coagulation-fibrinolysis balance in blood mononuclear cells and in gastric mucosa from patients with Helicobacter pylori infection.

INTRODUCTION: Helicobacter pylori and some of its virulence factors stimulate human blood mononuclear cells (MNC) in vitro to produce tissue factor (TF) and plasminogen activator inhibitor-2 (PAI-2). In this study we investigated the procoagulant-fibrinolytic potential of blood MNC in patients with H. pylori infection. In the same patients we also evaluated the coagulation-fibrinolysis profile in gastric tissue and in plasma. METHODS AND RESULTS: The production of TF and PAI-2 was evaluated in 61 patients with dyspepsia, 31 positive and 30 negative for H. pylori infection. TF expressed by MNC and PAI-2 accumulation in cell culture medium after incubation for 20 h at 37 degrees C were significantly higher in H. pylori(+) than in H. pylori(-) patients and were significantly correlated. TF and PAI-2 content in extracts of gastric mucosa was similar in the two groups whereas lower levels of tissue plasminogen activator (t-PA) and thrombomodulin (TM) antigens were found in the antrum of H. pylori(+) patients. No difference between the groups was observed in plasma thrombus precursor protein, prothrombin fragment 1+2, D-dimer, t-PA, PAI-1, TM and thrombin activatable fibrinolysis inhibitor. CONCLUSIONS: H. pylori infection is associated with functional abnormalities of blood MNC resulting in the coordinate expression of TF and antifibrinolytic activity. Changes in cell coagulation-fibrinolysis balance may represent a link between H. pylori infection and ischemic heart disease.

Fibrin down-regulates LPS- and PMA-induced tissue factor expression by blood mononuclear cells.

Several studies indicate that fibrin may play a functional role in inflammation by modulating a variety of cellular functions. We investigated the effect of fibrin on tissue factor (TF) production by blood mononuclear cells (MNC). Citrated human blood was recalcified and incubated at 37 degrees C for 1-4 h. The resulting clot was lysed by the addition of tissue plasminogen activator (t-PA) and MNC were isolated by density gradient centrifugation. A control blood sample was processed in the same way but omitting calcium addition and clot formation. Clot- and blood-derived MNC did not express detectable TF activity and antigen whatever the incubation time. Clot-derived MNC, however, generated on average 5 fold less TF (activity and antigen) than control cells, when stimulated with lipopolysaccharide (LPS, I microg/ml) for 3 h at 37 degrees C. A reduced TF response of clot-derived cells was also observed at mRNA level as indicated by RT-PCR and in situ hybridization. The effect was dependent on the incubation time within the clot, could not be reversed by enhancing LPS concentration or by adding serum, and was maintained if LPS was replaced by the tumor promoter PMA. A reduced TF response was also found when washed MNC were incorporated for 1 h at 37 degrees C within purified fibrin but not when the cells were incubated with fibrinogen, thrombin or fibrin split products alone. indicating that contact with fibrin was responsible for the inhibition of TF production. Fibrin-induced down-regulation of TF response to LPS and PMA by MNC may represent a negative feed-back aimed at limiting excessive blood clotting activation in immunoinflammatory diseases.

Angiotensin IV stimulates plasminogen activator inhibitor-1 expression in proximal tubular epithelial cells.

BACKGROUND: Angiotensin II (Ang II) has been shown to be implicated in the development of renal fibrosis in several forms of chronic glomerulonephritides, but the precise mechanisms of its effects remain unclear. It has recently been reported that Ang II stimulates the expression of plasminogen activator inhibitor-1 (PAI-1) in several cell lines. PAI-1 is a major physiological inhibitor of the plasminogen activator/plasmin system, a key regulator of fibrinolysis and extracellular matrix (ECM) turnover. PAI-1 induction by Ang II in endothelial cells seems to be mediated by Ang IV via a receptor that is different from Ang II type 1 and 2 receptors (AT1 and AT2). METHODS: In this study, we sought to evaluate the effects of Ang IV on PAI-1 gene and protein expression in a well-characterized and immortalized human proximal tubular cell line (HK2) by Northern blot and enzyme-linked immunosorbent assay. RESULTS: Ang IV stimulated PAI-1 mRNA expression, whereas it did not induce a significant increase in tritiated thymidine uptake after 24 hours of incubation. This effect was dose and time dependent. Ang IV (10 nM) induced a 7.8 +/- 3.3-fold increase in PAI-1 mRNA expression. The PAI-1 antigen level was significantly higher in conditioned media and the ECM of cells treated with Ang II and Ang IV than in control cells (both P < 0.02). Although Ang II induced a 4.2 +/- 2. 1-fold increase in PAI-1 mRNA expression, its effect underwent a dose-dependent reduction when amastatin, a potent inhibitor of the endopeptidases that catalyzes the conversion of Ang II to Ang IV, was added. In contrast, amastatin was not able to prevent the expression of PAI-1 mRNA induced by Ang IV. Finally, pretreatment of HK2 cells with losartan and N-Nicotinoyl-Tyr-N3-(Nalpha-CBZ-Arg)-Lys-His-Pro-Ile, the specific antagonists of AT1 and AT2 receptors, failed to modify PAI-1 mRNA expression as induced by Ang II. CONCLUSIONS: Our results demonstrate that Ang II stimulates PAI-1 mRNA expression and the production of its protein in human proximal tubular cells. This is mainly-if not exclusively-due to Ang IV, which acts on a receptor that is different than AT1 or AT2. Therefore, it can be hypothesized that the induction of PAI-1 by Ang IV may be implicated in the pathogenesis of renal interstitial fibrosis in several forms of chronic glomerulonephritides.

Prevention and therapy of experimental venous thrombosis in rabbits by desmin 370.

Desmin 370 (D370), a low molecular weight dermatan sulfate, has been shown to reduce the size of preformed thrombi in rats, via a mechanism largely independent of its anticoagulant activity. In the present study we investigated the therapeutic efficacy of D370 in rabbits with experimental jugular vein thrombosis. Experiments performed to evaluate the antithrombotic dosages in rabbits indicated that D370 prevented the formation of venous thrombi (Wessler model) in a dose-dependent manner with complete inhibition at 20 mg/kg. When injected to rabbits bearing a 30 min aged thrombus, D370 caused a time- and dose-dependent reduction in thrombus weight. Thrombi harvested 2 h after injection of 50 mg/kg of D370 were 71% smaller than thrombi from saline-treated rabbits and 50% smaller than pretreatment thrombi, suggesting a double effect of the drug: inhibition of thrombus accretion and reduction of the existing thrombus. Interestingly, pretreatment with the fibrinolytic inhibitor EACA (1 g/kg), significantly attenuated the therapeutic efficacy of D370, suggesting a possible involvement of the fibrinolytic system. Heparin (50 and 200 U/kg) was less active as therapeutic agent, the maximal decrease in thrombus weight, as compared to untreated rabbits, amounting to 38%. Heparin, moreover, caused a more pronounced prolongation of APTT than comparable antithrombotic dosages of D370. Our present data extend previous results on the therapeutic efficacy of D370 and underscore its potential as an alternative antithrombotic drug.

Effect of beta-carotene on the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat liver.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), is a rate-limiting enzyme in the biosynthesis of not only cholesterol but also a variety of non-sterol isoprenoids. It is subjected to multivalent feedback suppression by transcriptional and post-transcriptional control mechanisms mediated by sterols and non-sterol substances. In the present study, the effect of a plant isoprenoid, beta-carotene, on the expression of HMG-CoA reductase in rat liver was investigated. In control rats the hepatic levels of mRNA transcripts of HMG-CoA reductase increased following 2/3 partial hepatectomy with two peaks, one at 8 h and the other at 24 h. Administration of the carotenoid (70 mg/kg, given every alternate day for 3 consecutive weeks) partially inhibited the increase in the transcript level with a 50% reduction at 8 h and 30% reduction at 24 h post partial hepatectomy. Nuclear run-off assays with nuclei isolated from the resting liver and from livers of control rats and rats exposed to beta-carotene for 3 consecutive weeks and killed 8 h after partial hepatectomy indicated that beta-carotene did not inhibit the rate of transcription of HMG-CoA reductase gene. These observations suggest that beta-carotene regulates the expression of HMG-CoA reductase by some post-transcriptional mechanisms.

Genetic background and environment contribute synergistically to the onset of autoimmune diseases.

Autoimmune diseases result from the breakdown of "self" tolerance. Environmental factors appear to be responsible for triggering this errant immune response, directed against self-tissue determinants, only when a susceptible genetic background is present in an individual. Autoimmune diseases, normally characterized by their association with certain HLA alleles, also share other features: the presence of autoantibodies, autoreactive T lymphocytes, and an intermittent clinical course of exacerbations and remissions. In cases of organ-specific diseases, as well as in cases of multi-system autoimmune diseases, viruses are increasingly implicated as such environmental triggers. Current molecular biology techniques have permitted a fine dissection of the genetic background of susceptible individuals and have enabled a more complete characterization of the immunocompetent cells involved in this autoaggression. Molecular approaches will soon allow us to pinpoint the characteristics of the environmental stimuli, so that protective strategies could be formulated to spare susceptible individuals from their ill effects.

Similar patterns of hypomethylation in the beta-hydroxy-beta-methylglutaryl coenzyme A reductase gene in hepatic nodules induced by different carcinogens.

Our earlier studies demonstrated that the beta-hydroxy-beta-methylglutaryl coenzyme A (HMG CoA) reductase gene is hypomethylated and overexpressed in hepatic nodules initiated by 1,2-dimethylhydrazine (1,2-DMH). The study presented here examined whether the pattern of DNA methylation of the HMG CoA reductase gene in hepatic nodules reflected carcinogen-DNA interaction during initiation. Accordingly, hepatic nodules were generated in male Fischer 344 rats with either 1,2-DMH or aristolochic acid (AA), which interact predominantly with the guanine and adenine bases in DNA, respectively. DNA from individual nodules was restricted with HpaII, MspI, and HhaI, and the fragments obtained were hybridized to a cDNA probe for HMG CoA reductase. The results indicated that the hypomethylation pattern in the reductase gene in the nodules initiated with these two carcinogens was similar, although they interacted with different bases in the DNA. The question still remained whether the DNA fragments obtained by digesting the two sets of nodules with the restriction endonucleases were from the same domains in the genome of HMG CoA reductase. To examine this, probes for the different domains of the HMG CoA reductase gene were generated from the cDNA using the restriction enzyme Accl. Three probes were obtained: (i) a 5'-end fragment corresponding to the membrane-spanning region, (ii) a second fragment corresponding to the 3'-end of the protein, and (iii) a third fragment spanning the region between (i) and (ii). Each of these probes was radiolabeled and hybridized to the HpaII- and HhaI-generated fragments from the DNA of hepatic nodules initiated with 1,2-DMH and AA. Irrespective of the carcinogen used for initiation, hypomethylation occurred in all three domains of the gene. More important, the pattern of hypomethylation was similar in the nodules initiated by the two carcinogens. Furthermore, an overall similarity was seen in the hypomethylation patterns in the c-myc and c-Ha-ras genes in the DNA of nodules initiated with either 1,2-DMH or AA. These results raise the possibility that the pattern of hypomethylation established in the hepatic nodules may not directly reflect the interaction between the initiating carcinogen and DNA but may represent a unique phenotype of hepatic nodules.

Induction of hepatic nodules in the rat by aristolochic acid.

Aristolochic acid (AA), used as an anti-inflammatory agent in the past, is known to be mutagenic and carcinogenic to several organs of the rat, including forestomach, renal pelvis and urinary bladder. However, despite the induction of DNA adducts in the liver, no carcinogenic potential of AA has been reported in the latter organ. The present study was based on the rationale that the lack of carcinogenicity of AA to the liver could be because this chemical may not be necrogenic at the doses examined and liver cell proliferation has been established as an essential component for initiation of liver carcinogenesis in the rat. The results indicated that AA is non-necrogenic to the rat liver. However, a single non-necrogenic dose of AA (10 mg/kg b.w., i.p.) given 18 hours after 2/3 partial hepatectomy initiated liver cell carcinogenesis. The initiated cells are promotable with 1% dietary orotic acid, a liver tumor promoter, to form glutathione-S-transferase 7-7 positive hepatic foci and nodules.

DNA hypomethylation during liver cell proliferation induced by a single dose of lead nitrate.

DNA hypomethylation has already been found in regenerating rat liver and in hepatic (pre)malignant lesions when compared to normal non dividing liver. Here we report that extensive hypomethylation of hepatic DNA occurs in mitogen-treated rat liver. This effect can be seen as early as 12 h after metal treatment and parallels the liver dimension changes. Thus the lowering of the DNA 5-methylcytosine content appears to be a properly characteristic of cellular proliferation, independently from being caused by partial hepatectomy, carcinogen treatments or mitogen administration.

Transitory DNA hypomethylation during liver cell proliferation induced by a single dose of lead nitrate.

In the present study we have examined the effect of a single dose of the mitogen lead nitrate (75 mumols/kg body wt) on the methylation status of hepatic DNA in male Wistar rats. It was found that extensive hypomethylation of hepatic DNA occurs in mitogen-treated rat liver. This effect could be seen as early as 12 h after metal treatment and parallels the changes in liver weight. Probing with the methylation-sensitive enzymes HpaII, MspI, and HaeIII confirmed HPLC analyses and showed that methylation at these sites was affected by lead treatment. DNA hypomethylation has already been found in regenerating rat liver and in hepatic (pre)malignant lesions when compared to normal nondividing liver. Thus the lowering of the DNA 5-methylcytosine content appears to be a property characteristic of cellular proliferation, regardless of whether it is caused by partial hepatectomy, carcinogen treatments, or mitogen administration.

Effect of chemical carcinogens and partial hepatectomy on "in vivo" (35S)methionine interaction with rat liver tRNA.

The effect of carcinogens given by a single or multiple injections on the extent of (35S)methionine interaction with hepatic tRNA was studied in normal and partially hepatectomized rats. Either partial hepatectomy or administration of ethionine (100 or 330 mg/kg body weight) and dimethylnitrosamine (120 mg/kg body weight) by multiple i.p. injections inhibited the (35S)methionine-tRNA interaction, while administration of hepatocarcinogenic chemicals plus PH resulted rather in a stimulation. Methylnitrosourea enhanced the extent of interaction when administered in a single dose (100 mg per kg body weight) 18 h after partial hepatectomy.

"In vivo" (35S)methionine interaction with rat liver tRNA.

As part of a study to characterize the methionine role in tumorigenesis, we report that methionine sulfur interacts with rat liver tRNA "in vivo" (35S) radioactivity remained associated to the nucleic acid after a number of treatments, including tRNA deacylation. Similar data were obtained after administration of (methyl-3H) methionine, while no comparable tRNA labelling was detected when the aminoacid labelled in the aliphatic chain was given. Hplc analysis of (35S) tRNA enzymic hydrolysate showed two unidentified UV-absorbing radioactive peaks. NMR spectra of these two peaks did not reveal any thiomethyl group.