Antonio Angelini

Transforming growth factor-β1 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.

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 patient. In the present study, we investigared 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 compared to the classic reversing agent, Verapamil (Ver), used, as reference at 50 µM 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 µM). The mean percentage of remaining intracellular doxo were 55.4 ± 4.5 , 51.4 ± 3.9 and 50 ± 1.8 %, 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 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 increase in vivo, the efficacy of the anticancer treatment.

Transforming growth factor β1 induces IL-1 receptor antagonist production and gene expression in rat vascular smooth muscle cells

Abstract Atherosclerosis is an inflammatory-fibroproliferative process that may represent a possible milieu in which transforming growth factor- β (TGF- β ) can be involved. Vascular smooth muscle cells (VSMC) may represent a source or a target of a large number of growth factors and proinflammatory cytokines, including interleukin-1 and its receptor antagonist (IL-1Ra). We tested the effect of TGF- β 1 on IL-1Ra production and gene expression in rat VSMC cultures. We found a significant dose (3–30 ng/ml) and time-dependent (0–48 h) increase in IL-1Ra immunoactivity in the supernatant of conditioned medium and cell lysates. The maximal effect was observed with TGF- β at 30 ng/ml and after 24 h incubation time, respect to untreated cells (320±26 vs. 211±20 pg/ml; P β 1 induced an increased mRNA expression which began at 2 h and peaked at 18 h incubation time (about a 6-fold increase with respect to unstimulated cells). The effect of TGF- β 1 on IL-1Ra production was completely inhibited by an anti-IL-1 β antibody (10 μ g/ml) (from 320±81 to 181±46 pg/ml). These experiments suggest that TGF- β 1 , potentially produced in the vascular wall during atherogenesis, may play a pathophysiological role in the autocrine control of IL-1 actions, via VSMC IL-1Ra production.

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 μg/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 μg/ml; A260/A280 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 particulary suitable for a large scale screening for the diagnosis and prevention of the thrombotic risk.

New Method for the Extraction of DNA from White Blood Cells for the Detection of Common Genetic Variants Associated with Thrombophilia

In the last few years, FV Leiden, prothrombin 20210 and thermolabile methylene tetrahydrofolate reductase (MTHFR) 677 variants have been identified as possible risk factors associated with thrombophilia, and many PCR-based methods have been described for detecting these variations. However, the genomic DNA extraction is still a rate-limiting and time-consuming step in the PCR process. In an attempt to accelerate this procedure and make it suitable for routine laboratory, we report a single preparative technique for DNA extraction from peripheral blood samples using an anion-binding resin (GeneFizz). This method enables white blood cell lysis, DNA extraction and PCR amplification directly in the thermocycling tube on the thermocycler. The use of this new DNA extraction system coupled to a multiplex PCR allows rapid genetic screening of large cohorts of patients for thrombophilic risk factors.