Differential association between human prostacyclin receptor polymorphisms and the development of venous thrombosis and intimal hyperplasia: a clinical biomarker study.

OBJECTIVE AND METHODS: The role of prostacyclin in the development of venous thrombosis and vascular dysfunction in humans is unclear. In patients with deep vein thrombosis (DVT, n=34) and controls (matched for age, sex, indexes of systemic inflammation and metabolic status, n=20), we studied (i) differences on systemic markers of vascular disease and platelet activation and (ii) the influence of prostacyclin receptor gene (PTGIR) polymorphisms. MAIN RESULTS: Enhanced levels of urinary 11-dehydro-thromboxane (TX)B2 and plasma [soluble(s)] P-selectin, mostly platelet derived, were detected in DVT patients, whereas plasma von Willebrand factor levels and intima-media thickness of the common carotid arteries were not significantly different. In all patients' cohorts, we identified five PTGIR polymorphisms (three nonsynonymous: P226T, R212C, V196L; two synonymous: V53V, S328S). In the four individuals carriers of R212C polymorphism (three in DVT, one in controls), intima-media thickness values were significantly (P=0.0043) higher than those detected in individuals of all cohorts [1.68+/-0.38, 1.55 (1.4-2.2) vs. 1.05+/-0.33, 1.08 (0.01-1.68) mm, respectively, mean+/-SD, median (range)]. Moreover, enhanced sP-selectin and 11-dehydro-TXB2, in DVT versus controls, were statistically significant only in carriers of both synonymous PTGIR polymorphisms V53V/S328S. Only the PTGIR mutant R212C was dysfunctional when examined in an in vitro overexpression system. CONCLUSION: Our results suggest a propensity of enhanced platelet activation in DVT patients with PTGIR polymorphisms V53V/S328S. Moreover, we identified a dysfunctional PTGIR polymorphism (R212C) associated with intimal hyperplasia.

Modulatory effects of heparin on cellular accumulation and cytotoxicity of doxorubicin in MRP1-overexpressing HL60/doxo cells.

BACKGROUND: The overexpression of multidrug resistance protein (MRP1), associated with high levels of intracellular glutathione (GSH), is a well characterized mechanism of multidrug resistance (MDR) in several malignancies. Various chemosensitizers have been used in vitro to modulate the MRP1 activity, but the high toxicity limits their clinical application. Unfractionated heparin (UFH), is frequently used to prevent thrombo-embolic complications in cancer patients. This in vitro study aimed to elucidate the potential role of UFH as a sensitizer in anticancer clinical chemotherapy. MATERIALS AND METHODS: The human leukemic doxorubicin-resistant cell line (HL60/doxo), which overexpresses the MRP1 protein was treated with UFH alone or in combination with three different concentrations of doxo. The intracellular accumulation and cytotoxicity of doxo and the cellular GSH content were measured in comparison with the leukotriene LTD4 receptor antagonist, MK571, a specific MRP1 inhibitor. RESULTS: UFH increased doxo accumulation and cytotoxicity in the HL60/doxo cell line with respect to cells treated with doxo alone. UFH also decreased the cellular GSH content in the HL60/doxo cells with respect to the control, suggesting a potential involvement of UFH in doxo co-transport with GSH. CONCLUSION: Our results demonstrate that UFH modulates MRP1-mediated MDR in HL60/doxo cells expressing high MRP1 levels. These findings suggest a potential clinical application of heparin as an adjuvant to overcome MRP1-mediated drug resistance in cancer patients.

Reduced thromboxane biosynthesis in carriers of toll-like receptor 4 polymorphisms in vivo.

The recent demonstration that platelets express a functional toll-like receptor 4 (TLR4) prompted us to explore the influence of TLR4 polymorphisms (Asp299Gly alone or in combination with Thr399Ile) on thromboxane A(2) (TXA(2)) biosynthesis in vivo. In 17 subjects with TLR4 polymorphisms versus 17 wild type (untreated with aspirin, matched for age, sex, and cardiovascular risk factors), intima-media thickness in the common carotid arteries was significantly lower. Average urinary excretion of 11-dehydro-TXB(2), an index of systemic biosynthesis of TX, was significantly reduced by 65%. The urinary excretion of 2,3-dinor-6-keto-prostaglandin F(1alpha), an index of systemic biosynthesis of prostacyclin, was marginally depressed but the prostacyclin/TXA(2) biosynthesis ratio was significantly higher than in wild type. Selective inhibition of cyclooxygenase 2-dependent prostacyclin (by rofecoxib or etoricoxib) was associated with increased urinary excretion of 11-dehydro-TXB(2) in carriers of TLR4 polymorphisms, but not in wild-type, suggesting a restrainable effect of prostacyclin on platelet function in vivo in this setting. Reduced TXA(2) biosynthesis may contribute to the protective cardiovascular phenotype of TLR4 polymorphisms.

Inhibition of P-glycoprotein-mediated multidrug resistance by unfractionated heparin: a new potential chemosensitizer for cancer therapy.

Anticoagulant treatment with heparins is frequently used to prevent venous thromboembolism in cancer patients. In the present study, we investigated the ability of unfractionated heparin (UFH) to inhibit P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) on human breast cancer cell line (MDA-MB231) and its doxo-resistant subline. Results were a compared to the classic reversing agent, Verapamil (Ver), used, as reference at 50 microM concentration. We analysed the Pgp function by calcein acetoxymethylester (calcein-AM) uptake, a fluorescent marker substrate, before and after in vitro exposure to UFH at clinically achievable dose of 20 U/ml. The mean percentage of calcein-AM retained into cancer cells after 3 and 12 h were 32 +/- 10.9 and 45 +/- 12.3, respectively, for UFH pretreated cells and 25.3 +/- 8.7 and 29.4 +/- 10.4, respectively, for Ver pretreated cells when compared to control cells, receiving only medium. Pgp activity was studied by measuring intracellular drug accumulation in doxo-resistant subline, treated (2 h) with either UFH or Ver, prior exposure (2 h) at different doxo concentrations (2, 4 and 8 microM). The mean percentage of remaining intracellular doxo were 55.4 +/- 4.5 , 51.4 +/- 3.9 and 50 +/- 1.8 percent, respectively for UFH treated cells, and 44.1 +/- 5.8, 39.3 +/- 4.4 and 19.4 +/- 8.6%, respectively, for Ver treated cells as compared with control cells, receiving only doxo. These results were consistent with the increase of sensitivity to doxo of the same doxo-resistant subline resulting in a 2.2, 2.6 and 2.2-fold increase, respectively, for UFH-doxo combination and 2.2, 2.5 and 2.0-fold respectively, for Ver-doxo combination respect to cells receiving doxo alone, as assessed by MTT test. In conclusion, these findings demonstrate the potentiating effect in vitro of UFH on doxo accumulation and cytotoxicity in the MDA-231 cell line and its doxo-resistant subline and suggest that UFH could to be used, as an potential chemosensitizer, in clinical chemotherapy for increasing in vivo, the efficacy of the anticancer treatment.

Pravastatin up-regulates transforming growth factor-beta1 in THP-1 human macrophages: effect on scavenger receptor class A expression.

Statins have been shown to interact with several monocyte/macrophage functions. We tested the effect of pravastatin on transforming growth factor-beta1 (TGF-beta1) production and its possible involvement in scavenger receptors class A (SRA) expression in human THP-1 cells. TGF-beta1s biological activity in THP-1 cell conditioned medium, evaluated by luciferase activity of transfected cell with a TGF-beta responsive promoter, was increased in a dose-dependent manner after incubation with pravastatin (1-20 microM). Pravastatin (1-20 microM) induced a dose-dependent increase in TGF-beta1 mRNA expression and protein production in THP-1 cells. PMA-induced SRA gene and protein expression was suppressed by pravastatin with a mean 3-fold decrease at 10 microM. This last effect was reversed by a mouse monoclonal anti-TGF-beta1 neutralizing antibody. PD98059, a specific inhibitor of MAP kinase cascade, completely reversed pravastatin-induced SRA down-regulation. p44 and p42 isoforms showed a dose-dependent phosphorylation after treatment with pravastatin (1-20 microM) which was inhibited by a mouse monoclonal anti-TGF-beta1 antibody. Our results demonstrate that pravastatin significantly up-regulates TGF-beta1 expression which may be in involved in down-regulation of SRA expression in THP-1 cell cultures. A new pathway for pravastatin effects in atherogenesis can be suggested.

Identification of three genetic risk factors for venous thrombosis using a multiplex allele-specific PCR assay: comparison of conventional and new alternative methods for the preparation of DNA from clinical samples.

The demand for thrombophilia testing at the molecular level is increasing and with it the need for a simple and rapid and cost-saving procedure for the preparation of genomic DNA from whole blood samples. The aim of this paper is to compare the efficiency of two conventional commercial procedures (Genomic, Eurobio-Labtek, and Nucleospin, Macherey-Nagel) and two our alternative approaches (microwave irradiation and resin-binding method) for extraction DNA and their suitability and convenience for multiple sample preparation for simultaneous identification of the factor V Leiden, prothrombin 20210 and methylene tetrahydrofolate reductase (MTHFR) 677 variants by multiplex allele specific amplification (ASA-PCR). We have found that chemical-based kit (Genomic) produced higher DNA recovery (mean recovery 40 +/- 4.2 microg/ml; A260/A280 ratio 1,81 +/- 0.05) within 40 min., while the mini spin colum kit (Nucleospin Quickpure) obtained lower yield but the best DNA quality (mean recovery 25.7 +/- 2.3 microg/ml; A (260)/ A (280) ratio = 1,83 +/- 0.06) with fewer processing time (25 min). Costs of each extraction varied from 3.28 Euro for Genomic to 3.6 Euro for Nucleospin. Microwave radiation and resin-based method (GeneFizz) were single step/single tube procedures, that provided template DNA suitable for ASA-PCR assay, without any purification steps. The costs varied from 0.12 Euro for microwave to 1,23 Euro for resin based procedure. In conclusion, our alternative procedures were much faster (<15 min per extraction) and convenient (5.00-7.00 Euro per test) but equally sensitive compared to conventional DNA extraction methods. Moreover, these procedures are easily adaptable to the routine processing of high number of clinical samples and coupled with ASA-PCR assay result particularly suitable for a large scale screening for the diagnosis and prevention of the thrombotic risk.

Transforming growth factor-beta1 levels in hypertensive patients: association with body mass index and leptin.

BACKGROUND: Transforming growth factor-beta1 (TGF-beta1) has been demonstrated to be overexpressed in hypertension. Leptin, an adipocyte product, has been shown to play a role in obesity-related hypertension and in vitro studies demonstrated a biologic interaction between leptin and TGF-beta1. Thus, we evaluate a possible in vivo association between TGF-beta1, body mass index (BMI), and leptin circulating levels in hypertensive subjects. METHODS: Blood samples for fasting leptin and TGF-beta1, were evaluated in 29 overweight, 46 obese, and 29 nonobese hypertensive patients before and after a 12-week calorie-restricted diet. Monocyte cultures were used for in vitro experiments. RESULTS: Transforming growth factor-beta1 was significantly elevated in hypertensive obese patients (n = 46) as compared with TGF-beta1 levels of hypertensive patients with normal BMI (n = 29) (8. 9 +/- 3 ng/mL v 4.4 +/- 2; P < .001). The circulating levels of TGF-beta1 were associated with BMI and leptin levels in an univariate analysis (r = 0.59, P < .0001; r = 0.62, P < .0001, respectively) and these associations were still present after stepwise multivariate analysis. Weight loss of 10% produced a parallel decrease in TGF-beta1 (from 8.9 +/- 3 ng/mL to 5.3 +/- 2.8 ng/mL; P < .01) and leptin levels (from 30 +/- 24 ng/mL to 17 +/- 14; P < .05). In vitro experiments showed that leptin is able to induce a dose-dependent increase in TGF-beta1 production and mRNA expression in human monocyte cultures. CONCLUSIONS: Our data indicate that TGF-beta1 levels are positively associated with BMI and leptin levels in hypertensive patients and suggest that adipose tissue may be an important determinant of TGF-beta1 levels possibly by a leptin-dependent pathway.

Association of factor VII levels with inflammatory parameters in hypercholesterolemic patients.

Inflammatory markers have been demonstrated to be associated with increased risk of cardiovascular events. In this setting, C-reactive protein (CRP) was shown to add predictive value to cholesterol levels. We investigated hypercholesterolemic patients and related their inflammatory variables and their coagulation state focusing on factor VII, a coagulation protein which plays an established role in thrombogenesis. We examined the relationship between factor VII clotting activity (FVIIc), FVII antigen (FVIIAg) and activated FVII (FVIIa) levels against CRP, interleukin-6 soluble receptor (IL-6sR), P-selectin, soluble intercellular adhesion molecule-1 (ICAM-1) and transforming growth factor-beta(1) (TGF-beta(1)), in fifty-eight hypercholesterolemic subjects. Patients were subjected to 6-8 weeks of lipid lowering treatment with diet or diet plus pravastatin (40 mg/day). Univariate analysis showed that FVII levels were positively associated with CRP (FVIIAg: r=0.56, P<0.0001; FVIIc: r=0.57, P<0.0001; FVIIa: r=0.39, P<0.001) and IL-6sR (FVIIAg: r=0.59, P<0.0001; FVIIc: r=0.52, P<0.0001; FVIIa: r=0.47; P<0.001). CRP was still correlated, at the baseline, with FVIIAg and FVIIc levels after multiple stepwise regression analysis (FVIIAg: P<0.0001; FVIIc: P<0.0001, respectively) and with FVIIAg at the end of lipid lowering treatment (P<0.0001). Our data indicate that the FVII level is independently associated with inflammatory variables and suggest their pathophysiological link in hypercholesterolemic patients.

Increased transforming growth factor-beta(1) circulating levels and production in human monocytes after 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase inhibition with pravastatin.

OBJECTIVES: We sought to determine whether inhibition of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase with pravastatin affects transforming growth factor-beta(1) (TGF-beta(1)) circulating levels and its production in the monocytes of hypercholesterolemic patients. BACKGROUND: Transforming growth factor-beta(1) is a multifunctional growth factor/cytokine involved in many physiologic and pathologic processes, such as vascular remodeling and atherogenesis. Statins have been reported to have a modulatory role in cytokine expression in the monocytes of hyperlipidemic patients. METHODS: We evaluated, in a cross-over study design, plasma TGF-beta(1) levels and ex vivo TGF-beta(1) production in the monocytes of hypercholesterolemic patients before and after four to six weeks of lipid-lowering treatment with diet or diet plus 40 mg/day of pravastatin. In addition, isolated blood monocytes were subjected to pravastatin treatment and evaluated for TGF-beta(1) messenger ribonucleic acid (mRNA) expression and TGF-beta(1) in vitro production. RESULTS: Lipid-lowering treatment significantly decreased total cholesterol and low-density lipoprotein cholesterol plasma levels. Pravastatin, but not a low lipid diet, induced a significant increase in TGF-beta(1) plasma levels (from 1.7 +/- 0.5 ng/ml to 3.1 +/- 1.1 ng/ml, p < 0.001) and in ex vivo monocyte production (from 1.8 +/- 0.8 ng/ml to 3.9 +/- 1.0 ng/ml, p < 0.001). The increase in TGF-beta(1) levels was not related to the changes in the lipid profile observed with pravastatin. An increase of approximately twofold in TGF-beta(1) production and in mRNA expression was also observed after in vitro treatment of human monocytes with pravastatin (5 microM). Co-incubation with mevalonate reversed the in vitro effect of pravastatin. CONCLUSIONS: 3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibition with pravastatin increases TGF-beta(1) plasma levels, as well as monocyte production, in hypercholesterolemic patients. The mevalonate pathway plays a role in the regulation of TGF-beta(1) expression in human monocytes. A possible implication in the biologic and clinical effects of statins can be suggested.