Virtudes Vila

Circulating Endothelial Cells and Microparticles as Prognostic Markers in Advanced Non-Small Cell Lung Cancer

Background Circulating endothelial cells and microparticles have prognostic value in cancer, and might be predictors of response to chemotherapy and antiangiogenic treatments. We have investigated the prognostic value of circulating endothelial cells and microparticles in patients treated for advanced non-small cell lung cancer. Methodology/Principal Findings Peripheral blood samples were obtained from 60 patients before first line, platinum-based chemotherapy +/− bevacizumab, and after the third cycle of treatment. Blood samples from 60 healthy volunteers were also obtained as controls. Circulating endothelial cells were measured by an immunomagnetic technique and immunofluorescence microscopy. Phosphatidylserine-positive microparticles were evaluated by flow cytometry. Microparticle-mediated procoagulant activity was measured by the endogen thrombin generation assay. Results: pre- and posttreatment levels of markers were higher in patients than in controls (p<0.0001). Elevated levels of microparticles were associated with longer survival. Elevated pretreatment levels of circulating endothelial cells were associated with shorter survival. Conclusions/Significance Circulating levels of microparticles and circulating endothelial cells correlate with prognosis, and could be useful as prognostic markers in patients with advanced non-small cell lung cancer.

Influence of plasma and erythrocyte factors on red blood cell aggregation in survivors of acute myocardial infarction

Increased erythrocyte aggregation (EA) has been observed in patients with ischaemic heart disease (IHD), although most of these studies have been performed in the acute phase when reactant proteins may account for this increase. Little is known about the role played by the erythrocyte itself in this aggregation process. To ascertain the contribution of both plasma and erythrocyte factors to EA in IHD, we investigated the following parameters in 78 survivors of acute myocardial infarction (AMI) and in a well-matched control group of 98 subjects: EA, glucose, total cholesterol (T-Chol), low-density lipoprotein-cholesterol (LDL-Chol), high-density lipoprotein-cholesterol (HDL-Chol), triglycerides, apolipoproteins A(1) and B, protein and functional fibrinogen, plasma sialic acid, membrane sialic acid, and the cholesterol and phospholipid content of the erythrocyte membrane. AMI survivors showed higher glucose (p<0.001), a borderline increase in triglycerides (p = 0.043), and a statistical decrease in Apo A(1) (p= 0.003) relative to controls. EA, functional fibrinogen, and plasma sialic acid were statistically higher in AMI survivors than in controls (p= 0.001; p<0.001; p= 0.011, respectively). Membrane sialic acid content was statistically lower in AMI patients than in controls (p= 0.026). No differences were observed in either membrane cholesterol or phospholipids. Multivariate logistic regression analysis, in which EA was dichotomized as higher or lower than 8.7, demonstrated that triglyceride levels higher than 175 mg/dL (OR= 7.7, p= 0.001) and functional fibrinogen levels higher than 320 mg/dL (OR= 3.7, p= 0.004) were independently associated with a greater risk of erythrocyte hyperaggregability. Our results suggest that plasma lipids, predominantly triglycerides, and fibrinogen may not only enhance the development of ischaemic events by their recognized atherogenic mechanisms, but also by increasing EA.

A rapid method for isolation of fibrinogen from human plasma by precipitation with polyethylene glycol 6,000

Several techniques have been described for the isolation and purification of fibrinogen based on its precipitation properties. Tkse include precipitation with ethanol (11, with amino acids (2-4), using cationic detergents (S), and polyethylene glycol (6-8). However, most of these methods are tin-e-consuming and they produce a low yield, in sure cases, the final product is contamir&ed or partially denatured or degradated. The aim of our study is to develop a procedure for the isolation of fibrinfrom small volumes of human plasma, using polyethylene glycol 6,000 and saltin-out procedures, which is rapid and simple, produces high yields and purity, and is able to be used for the production of large amounts offikinqn.

Follow-up Study on the Utility of von Willebrand Factor Levels in the Diagnosis of Cardiac Allograft Vasculopathy

BACKGROUND Cardiac allograft vasculopathy (CAV) is the major cause of late death in patients undergoing heart transplantation (HT). The most validated method for its diagnosis is intravascular ultrasound (IVUS), and there are no sufficiently reliable non-invasive methods. von Willebrand factor (vWF) is a marker of endothelial dysfunction/activity that is rarely studied in the context of CAV. The purpose of this study was to determine whether patients with higher levels of vWF in the first year post-transplant will develop a greater degree of CAV. METHODS A prospective study of 113 consecutive cardiac transplant recipients was initiated in January 2002. vWF determinations were performed at 1, 2, 4, 6, 9 and 12 months post-transplant, at the same time as biopsies. Coronary arteriography and IVUS were performed on the first and last follow-up visits. Heart-lung transplants, retransplants and pediatric transplants were excluded from the study. Patients who died in the first month and those who refused consent were also excluded. The final analysis included 72 patients and 405 vWF determinations. CAV was defined as an intimal thickening of >or=0.5 mm on follow-up versus baseline IVUS. Patients with CAV (n = 41) and without CAV (n = 31) after 1 year of follow-up were compared. RESULTS Patients who developed CAV had a higher prevalence of prior dyslipidemia, ischemic heart disease as the cause of HT, and rate of rejection, as well as higher vWF levels (321 +/- 122 vs 243 +/- 100%, p < 0.05). The receiver-operator characteristic (ROC) curve showed that vWF values of 150% provided a sensitivity of 91%, and values of 400% a specificity of 91% (p < 0.0001). The variables associated with CAV in the multivariate analysis were prior dyslipidemia, rejections and vWF, both linearly and by groups. vWF levels of 300% to 400% increased the probability of developing CAV by 390%, and levels >400% by 500%, versus levels <200%. CONCLUSIONS vWF levels determined in the first year post-transplant help to distinguish a subgroup of patients with a higher incidence of CAV.

Erythrocyte membrane composition in patients with primary hypercholesterolemia

There are conflicting results regarding the erythrocyte membrane cholesterol and phospholipid content in patients with primary hypercholesterolemia (PHC), due to methodological problems in obtaining haemoglobin-free ghosts. At the same time, the different units used and the fact that the cholesterol and phospholipids are not expressed in relation with integral protein membrane content, produces contradictory results. We have analysed in 33 patients with PHC (12 male, 31 female) aged 43+/-12 years and in 33 healthy normolipaemic volunteers (9 male, 24 female) aged 43+/-13 years plasma lipids, along with, erythrocyte membrane cholesterol, phospholipids and integral proteins. PHC patients showed increased erythrocyte membrane cholesterol: 0.36+/-0.15 mg/mg when compared with controls: 0.29+/-0.75 mg/mg; p=0.018. Phospholipid membrane content, although higher in the cases, did not reach statistical significance (PHC patients: 0.38+/-0.15 mg/mg vs. 0.33+/-0.72 mg/mg; p=0.098). The cholesterol/phospholipids ratio (Chol/Ph) was 0.99+/-0.22 in PHC patients versus 0.92+/-0.28 in controls; p=0.127. Our results suggest that there is a slight increase in erythrocyte membrane cholesterol in patients with PHC. Given the increasing importance of erythrocyte membrane cholesterol in the stability of the atheroma plaque due its possible contribution to the clinical signs of ischaemic heart disease, it seems relevant to determine this parameter in risk populations. Therefore, a simple and reproducible method needs to be standardised which would enable comparisons between laboratories and facilitate further studies aimed to it as a marker of acute coronary syndromes.

Circulating Endothelial Cells and Procoagulant Microparticles in Patients with Glioblastoma: Prognostic Value

Aim Circulating endothelial cells and microparticles are prognostic factors in cancer. However, their prognostic and predictive value in patients with glioblastoma is unclear. The objective of this study was to investigate the potential prognostic value of circulating endothelial cells and microparticles in patients with newly diagnosed glioblastoma treated with standard radiotherapy and concomitant temozolomide. In addition, we have analyzed the methylation status of the MGMT promoter. Methods Peripheral blood samples were obtained before and at the end of the concomitant treatment. Blood samples from healthy volunteers were also obtained as controls. Endothelial cells were measured by an immunomagnetic technique and immunofluorescence microscopy. Microparticles were quantified by flow cytometry. Microparticle-mediated procoagulant activity was measured by endogen thrombin generation and by phospholipid-dependent clotting time. Methylation status of MGMT promoter was determined by multiplex ligation-dependent probe amplification. Results Pretreatment levels of circulating endothelial cells and microparticles were higher in patients than in controls (p<0.001). After treatment, levels of microparticles and thrombin generation decreased, and phospholipid-dependent clotting time increased significantly. A high pretreatment endothelial cell count, corresponding to the 99th percentile in controls, was associated with poor overall survival. MGMT promoter methylation was present in 27% of tumor samples and was associated to a higher overall survival (66 weeks vs 30 weeks, p<0.004). Conclusion Levels of circulating endothelial cells may have prognostic value in patients with glioblastoma.

Atorvastatin Neutralizes the Up-Regulation of Thrombospondin-1 Induced by Thrombin in Human Umbilical Vein Endothelial Cells

Statins have been reported to affect blood vessel formation. Thrombospondin-1 (TSP-1) is a multifunctional protein that affects vasculature systems such as platelet activation, angiogenesis, and wound healing. This study was designed to investigate the effect of atorvastatin on TSP-1 synthesis in thrombin-stimulated human umbilical vein endothelial cells (HUVECs), and its regulation by mevalonate or its derivatives. The results showed that atorvastatin down-regulated TSP-1 expression in HUVECs. This effect was fully reversed by mevalonate, farnesylpyrophosphate (FPP), and gerarylgeranylpyrophosphate (GGPP). Furthermore, farnesyltransferase and geranylgeranyltransferase inhibitors decreased TSP-1expression. It was also found that thrombin increased TSP-1 expression in HUVECs. Atorvastatin (0.1, 1, and 10 muM) decreased TSP-1 in thrombin-stimulated cells (45%, 66%, and 80%). Mevalonate partially reversed this inhibitory effect of atorvastatin on TSP-1, whereas the presence of FPP and GGPP did not alter TSP-1. Rho-kinase inhibitor neutralized the up-regulation of TSP-1 induced by thrombin. In conclusion, atorvastatin inhibits TSP-1 expression in endothelial cells via the mevalonate pathway. Rho protein activation is necessary for up-regulation of TSP-1 synthesis induced by thrombin. Because FPP and GGPP are essential for the activity of Rho proteins, inhibition of these proteins may constitute the mechanism by which atorvastatin inhibits thrombin up-regulated TSP-1 expression.

Study of the formation of fibrin clot in cirrhotic patients. An approach to study of acquired dysfibrinogenemia

Alterations in the coagulation system are common in patients with liver disease. We have examined the importance of the species and chains of fibrinogen in 3 groups of cirrhotic patients. The study of the gelation of fibrinogen in cirrhotic patients shows that the lag time increases in 80.3% of them and that the maximum gelation rate is altered in 51% of these plasmas. Also it is observed that 80% of the plasmas from cirrhotic patients have a percentage (23.3 +/- 7.7%) of unpolymerized alpha chain, after highly cross-linked fibrin formation. These alterations, in lag time and in the maximum gelation rate, have no significant correlation with the situation of the fibrinolytic system in these patients. The study of isolated fibrinogen from cirrhotic patients and normal subjects plasma, shows that there are no objective alterations in the percentage of fibrinogen species, the amount of sialic acid or the ratio of polypeptide chains.

Isolation of fibrin(ogen) degradation products from normal plasma by affinity chromatographic study.

An attempt was made to develop a method to isolate directly the fibrinogenfibrin (FDP and/or fdp) degradation products from plasma by means of small chromatographic columns of activated Sepharose 4-B coupled with antifibrinogen serum. The study of the material adsorbed was performed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (PAGE-SDS). Four electrophoretic bands with antigenic capacity against antifibrinogen serum were observed. The study of their molecular weights, of their polypeptide composition after reduction, and of their immunological response against antisera anti-D and anti-E allowed their identification as fibrinogen, D-dimer fragment, and fragment E, respectively. The possibility of using this technique for the differential diagnosis between a primary fibrinogenolysis and a secondary fibrinolysis in a thrombotic process is suggested, as well as its use in the control of thrombolytic therapy.

Hypercoagulable state after thrombolytic therapy in patients with acute myocardial infarction (AMI) treated with streptokinase

Fibrinogen activity was studied in 70 patients with AMI who were treated with an intravenous infusion of SK (800,000 U/30 min or 1.5 mill U/60 min). Patients received a continuous infusion of heparin after thrombolytic therapy was completed. 800,000 U and 1.5 mill U SK recanalized infarct-related arteries at a rate of 78%. Early re-infarction occurred in 6% in each group. Upon admission to the hospital patients showed a hypercoagulable state that may be related to an elevated level of fibrinogen and HMW fibrinogen (70.5 +/- 2 vs 65 +/- 2% in patient and normal plasmas, respectively) that changed to a transitory hypercoagulable state indicated by decreased fibrinogen levels after SK treatment. Forty-eight hours after SK, a new fibrinogen hyperfunction, related to an increase in fibrinogen level and especially HMW synthesized fibrinogen (82 +/- 1 or 81 +/- 1%, 800,000 and 1.5 mill U SK, respectively) was observed, which was neutralized by heparin therapy (1,660 U/h with continuous infusion). The elevated levels of fibrinogen (363 +/- 21 vs 240 +/- 8 mg/dl in patient and normal plasmas, respectively) and HMW fibrinogen (70 +/- 3% with both SK hypercoagulable state that is not neutralized by the heparin dose were compared with those whose arteries recanalized. The former group had a higher concentration of fibrinogen (197 +/- 31 vs 147 +/- 18 mg/dl), HMW fibrinogen (78 +/- 0.5 vs 74 +/- 0.3%, respectively), and FPA (130 +/- 3 vs 6 +/- 4 pmol/ml) and more extensive fibrin gel formation kinetics (gelation rate 3.3 +/- 1.4 vs 1.1 +/- 0.2 OD/s x 10(-4), respectively) than the second group. The hypercoagulable state found in patients with acute myocardial infarction undergoing thrombolytic therapy may be related mainly to the progression of HMW fibrinogen and fibrinogen levels.