Vicenta Martínez-Sales

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.

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.

Association of the Thrombomodulin Gene c.1418C>T Polymorphism With Thrombomodulin Levels and With Venous Thrombosis Risk

Objective—To investigate the association of the THBD c.1418C>T polymorphism, which encodes for the replacement of Ala455 by Val in thrombomodulin (TM), with venous thromboembolism (VTE), plasma soluble TM, and activated protein C levels. In addition, human umbilical vein endothelial cells (HUVEC) isolated from 100 umbilical cords were used to analyze the relation between this polymorphism and THBD mRNA and TM protein expression. Approach and Results—The THBD c.1418C>T polymorphism was genotyped in 1173 patients with VTE and 1262 control subjects. Levels of soluble TM and activated protein C were measured in 414 patients with VTE (not on oral anticoagulants) and 451 controls. HUVECs were genotyped for the polymorphism and analyzed for THBD mRNA and TM protein expression and for the ability to enhance protein C activation by thrombin. The 1418T allele frequency was lower in patients than in controls (P<0.001), and its presence was associated with a reduced VTE risk, reduced soluble TM levels, and increased circulating activated protein C levels (P<0.001). In cultured HUVEC, the 1418T allele did not influence THBD expression but was associated with increased TM in cell lysates, increased rate of protein C activation, and reduced soluble TM levels in conditioned medium. Conclusions—The THBD 1418T allele is associated with lower soluble TM, both in plasma and in HUVEC-conditioned medium, and with an increase in functional membrane–bound TM in HUVEC, which could explain the increased activated protein C levels and the reduced VTE risk observed in individuals carrying this allele.

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.

Regulation of cytosolic PlA2 activity by PP1/PP2A serine/threonine phosphatases in human platelets

Platelet thromboxane A2 (TXA2) synthesis is an important pathway of platelet reactivity. We report that in thrombin-stimulated platelets, PP1/PP2A serine/threonine phosphatases regulate phospholipase A2 (cPLA2) activity, which is required for TXA2 synthesis. Two mechanisms are involved: (a) constitutively active PP1/PP2A regulate cPLA2 phosphorylation, and (b) PP1/PP2A activity mediates agonist-induced increase in cytosolic Ca2+ ([Ca2+]i). Inhibition of PP1/PP2A with okadaic acid (OA) induces cPLA2 phosphorylation but reduces Ca2+ responses: release from intracellular stores and influx through the plasma membrane, particularly that mediated by store-mediated Ca2+ entry (SMCE). A significant correlation (r = 0.64) exists between OA-regulated [Ca2+]i and TXA2 synthesis. Okadaic acid-induced decrease in SMCE and the associated TXA2 synthesis are mediated by a reduction in protein-tyrosine phosphorylation. This reduction is not due to inhibition of tyrosine kinases but rather to an OA-mediated increase in tyrosine phosphatases. This is the first study to report that PP1/PP2A phosphatases are involved in the regulation of the two key elements in eicosanoid synthesis, [Ca2+]i and cPLA2 phosphorylation. Moreover, PP1/PP2A regulation of [Ca2+]i and tyrosine phosphorylation may be important for other calcium-dependent processes and/or signal transduction mechanisms in platelets.

Circulating endothelial and endothelial progenitor cells in non-small-cell lung cancer

New treatments have recently been introduced for treating non-small-cell lung cancer. Chemotherapeutic agents, such as pemetrexed, and targeted therapies, such as bevacizumab, erlotinib or gefitinib, have extended treatment options for selected histological subgroups. Antiangiogenic treatments, either associated with conventional chemotherapeutic drugs or given alone as maintenance therapy, constitute an active clinical research field. However, not all lung cancer patients benefit from antiangiogenic compounds. Moreover, tumour response assessment is often difficult when using these drugs, since targeted therapies generally do not cause rapid and measurable tumour shrinkage but, rather, long stabilisations and slight density changes on imaging tests. The finding of clinical or biological factors that might identify patients who will better benefit from these treatments, as well as identifying surrogate markers of tumour response and prognosis, is an issue of great interest. In that sense, different research lines have investigated the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor receptor (VEGFR) pathways. Circulating endothelial (CECs) and endothelial progenitor cells (CEPCs) are of prognostic value in different types of cancers, and relevant data are published about their potential usefulness as predictors of response to chemotherapy and antiangiogenic treatments. In this review, we discuss the data available on the role of CECs and CEPCs as prognostic factors and as surrogate markers of treatment response in non-small-cell lung cancer.

Increased circulating endothelial cells and microparticles in patients with psoriasis

INTRODUCTION Psoriasis is a chronic pathology characterized by increased inflammation that can be associated with changes in the vascular endothelium. We quantified the levels of circulating endothelial cells (CECs) and microparticles (MPs) in patients with psoriasis in order to analyze their relationship with endothelial and inflammation markers, subclinical atherosclerosis and microcirculation. METHODS We studied 20 patients and 20 controls. Circulating markers of endothelial damage (CEC, MPs and von Willebrand factor, vWF) and inflammation (E-selectin, E-sel; Interleukin-6, IL-6 and C-reactive protein, CRP) were determined. Subclinical atherosclerosis was assessed by carotid ultrasound to obtain intima-media thickness. Microcirculation was evaluated by nailfold capillaroscopy. RESULTS CECs, MPs, vWF, CRP and E-sel levels were significantly elevated in patients when compared with controls (p <  0.05). Ninety-four and fifty-three percentage of patients had CEC and MP levels higher than 99th percentile in controls. Forty-seven percent of patients simultaneously showed increased CEC and MP levels. MPs correlate with the inflammatory markers and with the intima-media thickness. CECs correlate with the capillaries loops per mm (p <  0.05). CONCLUSION Psoriasis patients show elevated CECs and MPs, as a sign of endothelial dysfunction, which correlates with inflammatory markers as well as subclinical atherosclerosis and some capillaroscopy findings.

Circulating Endothelial Cells in Patients with Heart Failure and Left Ventricular Dysfunction

Introduction and Aims: Acute and chronic heart failure may manifest different degrees of endothelial damage and angiogenesis. Circulating endothelial cells (CEC) have been identified as marker of vascular damage. The aim of our study was to evaluate the evolution of the CEC at different stages of patients with heart failure. We also investigated a potential correlation between CEC and markers of vascular damage and angiogenesis. Methods: We studied 32 heart failure patients at hospital admission (acute phase) and at revision after 3 months (stable phase) and 32 controls. Circulating markers of endothelial damage (CEC; von Willebrand factor, vWF and soluble E-selectin, sEsel) and angiogenesis (vascular endothelial growth factor, VEGF and thrombospondin-1) were quantified. Results: Levels of CEC, vWF, sEsel and VEGF are significantly higher in heart failure patients than in controls. Levels of CEC (36.9 ± 15.3 vs. 21.5 ± 10.0 cells/ml; p < 0.001), vWF (325 ± 101 vs. 231 ± 82%; p < 0.001) and VEGF (26.3 ± 15.2 vs. 21.9 ± 11.9 ng/ml; p < 0.001) are significantly higher in the acute phase than in the stable phase of heart failure. CEC levels correlate with vWF and VEGF. Results show than 100% of patients in acute phase and 37.5% in stable phase have levels of CEC higher than the 99th percentile of the distribution of controls (16 cells/ml). Therefore, increases in CEC represent a relative risk of 9.5 for heart failure patients suffering from acute phase. Conclusions: CEC, in addition to being elevated in heart failure, correlate with vWF levels, providing further support for CEC as markers of endothelial damage. Levels of CEC are associated with the acute phase of heart failure and could be used as a marker of the worsening in heart failure.