Ettore Porreca

Association between enhanced soluble CD40L and prothrombotic state in hypercholesterolemia : Effects of statin therapy

Background—Hypercholesterolemia is associated with inflammation and the prothrombotic state. CD40-CD40 ligand (CD40L) interactions promote a prothrombotic response in nucleated cells. The aim of this study was to characterize the in vivo expression of soluble CD40L (sCD40L) in hypercholesterolemia, to correlate it with the extent of the prothrombotic state, and to investigate whether it may be modified by statins. Methods and Results—We studied 80 hypercholesterolemic patients and 80 matched healthy subjects. Hypercholesterolemic subjects had enhanced levels of sCD40L, factor VIIa (FVIIa), and prothrombin fragment 1+2 (F1+2) compared with healthy subjects. sCD40L correlated with total cholesterol and LDL cholesterol. Moreover, sCD40L was positively associated with in vivo platelet activation, as reflected by plasma P-selectin and urinary 11-dehydro-thromboxane B2, and with procoagulant state, as reflected by FVIIa and F1+2. Inhibition of cholesterol biosynthesis by pravastatin or cerivastatin was associated with comparable, significant reductions in sCD40L, FVIIa, and F1+2. Conclusions—This study suggests that sCD40L may represent the molecular link between hypercholesterolemia and the prothrombotic state and demonstrates that statin therapy may significantly reduce sCD40L and the prothrombotic state.

The haematocrit and platelet target in polycythemia vera

Polycythemia vera (PV) is a chronic myeloproliferative disorder whose major morbidity and mortality are thrombohaemorragic events and progression to acute leukaemia or myelofibrosis. Whether the haematocrit and platelet count predict such complications remains unclear. The European Collaboration on Low‐dose Aspirin in Polycythemia Vera prospective study included 1638 PV patients. A total of 164 deaths (10%), 145 (8·85%) major thrombosis and 226 (13·8%) total thrombosis were encountered during 4393 person‐years follow‐up (median 2·8 years). In time‐dependent multivariable analysis, a haematocrit in the evaluable range of 40–55% was neither associated with the occurrence of thrombotic events, mortality nor with haematological progression in the studied population. The haematocrit of patients in the highest and lowest deciles at baseline was maintained within a narrow interval of haematocrit values ranging from 40% to 47% throughout follow‐up. High platelet count was associated with a lower progression rate to acute leukaemia/myelofibrosis, whereas it had no significant relationship with thrombotic events or mortality. Our findings do not suggest that the range of haematocrit (<55%) and platelet counts (<600 × 109/l) we encountered in our population had an impact on the outcome of PV patients treated by current therapeutic strategies.

Aortic antioxidant defence mechanisms: time-related changes in cholesterol-fed rabbits

In 24 rabbits fed a hyperlipidic diet (0.5% cholesterol, 5% lard and 5% peanut oil) for 10 (group A1), 30 group B1) and 60 days, (Group C1), compared to 24 control rabbits fed a standard diet for the same periods, antioxidant defence system (total superoxide dismutase, catalase, total thiol compounds selenium-dependent and selenium-independent glutathione peroxidase, glutathione reductase, glutathione transferase) and lipid peroxidation (thiobarbituric acid-reactive substances) in the aortic wall were tested. The percent of intima with grossly apparent atherosclerosis, is assessed by staining with the lipophilic dye Sudan IV, was negligible in group A1, but increased progressively in groups B1 (22.7-6.7%) and C1 (56.8-8.8%). Compared to the controls, a significant rise in superoxide dismutase activity was observed after 30 days of hyperlipidic diet, with a further marked increase at 60 days. Total thiol compounds and selenium-dependent glutathione peroxidase activity rose progressively from 10 to 30 and 60 days in cholesterol-fed rabbits. On the contrary, catalase, glutathione reductase and glutathione transferase activities significantly decreased in all experimental groups. Selenium-independent glutathione peroxidase activity was not detectable. Thiobarbituric acid-reactive substances increased about 3 times in hyperlipidemic rabbits. In conclusion, the changes in aortic antioxidant defence mechanisms and lipid peroxidation precede the massive vascular lipid infiltration in cholesterol-fed rabbits; some antioxidant mechanisms are stressed (superoxide, dismutase, glutathione peroxidase, total thiol compounds), whereas others are depressed (catalase, glutathione reductase, and glutathione transferase), thus potentially reducing or increasing vascular susceptibility to oxidative injury.

Accuracy of diagnostic tests for clinically suspected upper extremity deep vein thrombosis: a systematic review

Summary.  Background: The best available test for the diagnosis of upper extremity deep venous thrombosis (UEDVT) is contrast venography. The aim of this systematic review was to assess whether the diagnostic accuracy of other tests for clinically suspected UEDVT is high enough to justify their use in clinical practise and to evaluate if any test can replace venography. Methods: MEDLINE and EMBASE databases were searched from inception to June 2009. Two reviewers independently evaluated study eligibility, extracted data, and assessed study quality. Results: We identified 17 papers, reporting on 793 patients. Overall, the methodological quality was poor, sample sizes were small, and large between‐study differences were observed in spectrum and design. The summary estimates of sensitivity (95% confidence interval) were 97% (90–100%) for compression ultrasonography, 84% (72–97%) for Doppler ultrasonography, 91% (85–97%) for Doppler ultrasonography with compression, and 85% (72–99%) for phleboreography. The corresponding summary estimates of specificity were, respectively, 96% (87–100%), 94% (86–100%), 93% (80–100%), and 87% (71–100%). Clinical findings, a clinical score, D‐dimer, magnetic resonance imaging, rheography and plethysmography were evaluated in one study each, involving a median number of 46 patients (range 21–214). Sensitivity and specificity ranged from 0% to 100% and from 14% to 100%. Conclusions: Methodological limitations, large between‐study differences and small sample sizes limit the evidence of tests for clinically suspected UEDVT. Compression ultrasonography may be an acceptable alternative to venography. The addition of (color) Doppler does not seem to improve the accuracy. Adequately designed studies are warranted to confirm these findings.

Expression of interleukin 8 (IL-8) and substance P in human chronic pancreatitis

BACKGROUND Changes in substance P content and a relationship between the degree of perineural inflammation and pain has been demonstrated in chronic pancreatitis. Whether a relationship exists between neural alteration and pancreatic inflammation (neurogenic inflammation) is not known. AIMS In the present study we evaluated gene expression of preprotachykinin A (PPT-A), the gene encoding substance P, and interleukin 8, a proinflammatory and hyperalgesic mediator whose release is co-regulated by substance P. PATIENTS Pancreatic tissue specimens obtained from 21 patients (16 male, five female) with chronic pancreatitis and 18 healthy organ donors (nine male, nine female) were analysed. METHODS Gene expression of PPT-A and interleukin 8 was studied by northern blot analysis. Respective proteins were localised using immunohistochemistry. RESULTS Northern blot analysis showed that PTT-A mRNA expression levels were present at comparable levels in normal and chronic pancreatitis tissue samples. In contrast, interleukin 8 mRNA was expressed at very low levels in normal controls but was increased 41-fold (p<0.001) in chronic pancreatitis tissue samples. Using immunohistochemistry, interleukin 8 protein was localised mainly in immune cells often found around enlarged pancreatic nerves. In addition, in chronic pancreatitis, intense interleukin 8 immunostaining was present in metaplastic ductal cells of the atrophic pancreatic parenchyma. In chronic pancreatitis samples there was a positive relationship between interleukin 8 mRNA levels and the presence of ductal metaplasia (r=0.795; p<0.001) and the inflammation score (r=0.713; p<0.001). CONCLUSIONS Our data indicate that in chronic pancreatitis, the increase in substance P in enlarged pancreatic nerves is not caused by enhanced intrapancreatic PTT-A mRNA expression, suggesting that the location of substance P synthesis is outside of the pancreas. In addition, localisation of interleukin 8 positive immune cells around pancreatic nerves further supports the existence of neuroimmune interactions as a pathophysiological mechanism in chronic pancreatitis.

Increased Expression of Transforming Growth Factor-β1 as a Stabilizing Factor in Human Atherosclerotic Plaques

Background and Purpose— Transforming growth factor-β (TGF-β) is a growth factor/cytokine involved in vascular remodeling and atherogenesis. Recent studies in apolipoprotein E-deficient mice have demonstrated a pivotal role of TGF-β in the maintenance of the balance between inflammation and fibrosis in atherosclerotic plaques. Furthermore, inhibition of TGF-β signaling has been shown to accelerate plaque formation and its progression toward an unstable phenotype in mice. However, if this mechanism is operative also in humans is still unknown. The aim of this study was to characterize the expression of TGF-β1 in human carotid plaque and to correlate it with the extent of inflammatory infiltration and collagen content with the clinical signs of plaque instability. Methods— Plaques were obtained from patients undergoing carotid endoarterectomy and divided into symptomatic and asymptomatic according to clinical evidence of recent transient ischemic attack or stroke. Plaques were analyzed for TGF-β1 expression by Immunocytochemistry, Western, and Northern blotting analysis. Immunocytochemistry was used to identify CD68+ macrophages, CD3 T lymphocytes, HLA-DR+ cells, and α-smooth muscle cells. Procollagen and interstitial collagen content were analyzed by immunohistochemistry and Sirius Red staining, respectively. Results— Plaque TGF-β1 mRNA was increased up to 3-fold in asymptomatic as compared with symptomatic plaques. Plaques from asymptomatic group had fewer (P<0.0001) macrophages and T lymphocytes compared with symptomatic plaques. TGF-β1 gene was transcriptionally active as demonstrated by increased (P<0.0001) TGF-β1 protein expression in asymptomatic plaques. Immunohistochemistry showed that TGF-β was mainly expressed in plaque shoulder and was associated with a comparable increase (P<0.0001) in plaque procollagen and collagen content. Conclusions— In conclusion, this study demonstrates the higher expression of TGF-β1 in human asymptomatic lesions and provides evidence that TGF-β1 may play an important role in the process of plaque stabilization.

Monocyte Chemotactic Protein 1 (MCP-1) Is a Mitogen for Cultured Rat Vascular Smooth Muscle Cells

The involvement of inflammatory mechanisms in the progression of atherosclerosis has recently been suggested. Monocyte chemotactic protein 1 (MCP-1) is a soluble protein which is implicated in acute and chronic inflammatory processes, including atherosclerosis. We evaluated the effect of human recombinant MCP-1 on the in vitro proliferation of rat vascular smooth muscle cells (VSMCs). Incubation of VSMCs with MCP-1 (50-200 ng/ml) in the presence of 0.5% FCS significantly increased cell proliferation, [3H]-thymidine incorporation and the proliferative S fraction, measured by flow cytometry, compared to control cells. The proliferative effect of MCP-1 was specific, as shown by inhibition with a rabbit polyclonal serum to MCP-1. Moreover, the mitogenic effect of MCP-1 was significantly inhibited by downregulation of protein kinase C (PKC) activity and by incubation with H-7, a protein kinase inhibitor, suggesting the involvement of the PKC system. Verapamil, a Ca2+ channel blocker, also reduced the stimulatory effect of MCP-1 on cell proliferation. This study demonstrates that MCP-1 does not merely have a chemotactic activity, but also a mitogenic effect on cultured rat VSMCs.

Incidental venous thromboembolism in ambulatory cancer patients receiving chemotherapy.

While the association between cancer and symptomatic venous thromboembolism (VTE) is well established, the incidence and risk factors for incidental VTE in cancer patients remain unclear. The medical records of 1,921 consecutive cancer patients starting chemotherapy from January 2003 up to March 2009 were identified. Patients with a positive history of VTE were excluded. Pre-existing signs of VTE, kind and stage of malignancy, first and subsequent lines of chemotherapy, and all follow-up computed tomography (CT) scans were analysed. The primary outcome was incidental VTE. Overall, there were 101 (5.3%) VTE, 62 (3.2%) incidental and 39 (2.0%) symptomatic during a median of eight months (range 3-72). The incidence on CT scans was 0.58% (95%CI: 0.44-0.74). Incidental VTE included 24 pulmonary embolism, 28 deep venous thrombosis of the extremities, and 10 thromboses of the cava or splanchnic veins. Half of the incidental VTE occurred in the first 3-6 months of chemotherapy with a relatively higher incidence in gynecological and lung cancers. The presence of metastases, high leukocyte count, and platin-based chemotherapy increased the risk up to three-fold. All patients with incidental VTE regardless the location received half to full therapeutic doses of low-molecular-weight heparin for a minimum of three months. In summary, incidental VTE is a relative common finding in patients with solid tumours, especially in the first months of chemotherapy. Further research is needed to understand the natural history of incidental thrombosis in order to develop adequate management guidelines.

Leptin induces tissue factor expression in human peripheral blood mononuclear cells: a possible link between obesity and cardiovascular risk?

Summary.  Background: Obesity is a major modifiable risk factor for cardiovascular disease. Leptin, the hormone synthesized and released primarily by adipose tissue and found increased in obese individuals, has been implicated in the regulation of inflammation and arterial and venous thrombosis. Objective: To investigate the role of tissue factor (TF), the pivotal agonist of the clotting cascade, as a link between obesity and cardiovascular disease. Methods and results: In 15 obese patients, plasma levels of leptin and TF as well as TF expression in resting and endotoxin‐stimulated mononuclear leukocytes (MN) were increased when compared with healthy donors. In a selected sample of obese patients, loss of body weight led to decreased circulating leptin levels, accompanied by a reduction in plasma TF as well as in TF expression, both in resting and endotoxin‐stimulated MN. In subsequent in vitro experiments, leptin was incubated with MN from healthy subjects. Leptin induced TF activity and antigen in a dose‐dependent fashion, as assessed by clotting assay and ELISA, respectively. Increased migration of c‐Rel/p65 into the nucleus, as determined by EMSA, and development of TF mRNA in monocytes, as assessed by RT‐PCR, were observed. Experiments with mitogen‐activated protein kinase (MAPK) inhibitors, indicated the involvement of p38 and ERK1/2 pathways. Conclusions: The presence of TF‐expressing MN in blood from obese subjects and the in vitro induction of TF by pharmacologic concentrations of leptin in MN from healthy subjects suggest that TF expression by leptin‐stimulated monocytes may contribute to the cardiovascular risk associated with obesity.

Antiproliferative effect of desferrioxamine on vascular smooth muscle cells in vitro and in vivo.

Vascular smooth muscle cell (VSMC) growth is a primary component of accelerated and spontaneous atherosclerosis. Previous studies have shown that iron may be involved in the control of enzymatic activities that modulate DNA synthesis in human cells. In this study the effects of the iron chelator desferrioxamine on in vitro and in vivo VSMC proliferation were tested. Rat VSMCs in culture and a rabbit model of carotid artery balloon injury were used. Desferrioxamine showed a significant inhibitory effect on [3H]thymidine incorporation in cell cultures that was antagonized by iron supplementation. Desferrioxamine also provided effective preventive myointimal VSMC proliferation as assessed by bromodeoxyuridine labeling and morphometric analysis of endoluminal stenosis. These experiments suggested that iron may be involved in the control of VSMC proliferation and that desferrioxamine may have a role in preventing VSMC growth and myointimal proliferative lesions.