Impact of sample processing on the measurement of circulating microparticles: storage and centrifugation parameters.

BACKGROUND: Microparticles (MPs) have been shown to be markers of cellular activation and interactions. Pre-analytical conditions such as the centrifugation protocol and sample storage conditions represent an important source of variability in determining MPs values. The objectives of this study were to evaluate the influence of sample storage conditions and centrifugation speed and temperature on the determination of MPs in plasma. METHODS: Citrate-anticoagulated blood samples obtained from 21 healthy subjects were centrifuged under four different protocols involving different speeds (2500 g or 1500 g) and temperatures (4 °C or 20 °C) to isolate platelet-poor plasma (PPP). The number of MPs in fresh and frozen-thawed PPP were analyzed by flow cytometry, and MPs-mediated procoagulant activity was determined by a thrombin generation test and phospholipid-dependent procoagulant tests. RESULTS: The number of MPs and their procoagulant activity were affected by freeze-thaw cycling and centrifugation speed but not by centrifugation temperature. Sample freezing increased MPs number (six-fold) and thrombin generation (four-fold), and decreased clotting time (two-fold). Low centrifugation speed caused an increase in MPs number and a parallel increase in MP-mediated procoagulant activity. CONCLUSIONS: Sample storage conditions and centrifugation speed are important processing conditions affecting MPs number and activity. Before any study, the protocol for MPs isolation should be optimized to ensure a reliable characterization of MPs, which could provide important information for diagnostic purposes and for understanding the pathogenesis of diseases.

Phase II trial of irinotecan and metronomic temozolomide in patients with recurrent glioblastoma.

This phase II study was conducted to determine the efficacy and safety of metronomic temozolomide (TMZ) in combination with irinotecan in glioblastoma (GB) at first relapse. Patients with GB at first relapse received TMZ 50 mg/m/2day divided into three doses, except for a single 100 mg/m2 dose, administered between 3 and 6 h before every irinotecan infusion. Irinotecan was given intravenously at the previously established dose of 100 mg/m2 on days 8 and 22 of 28-day cycles. Treatment was given for a maximum of nine cycles or until progression or unacceptable toxicity occurred. Vascular endothelial growth factor and its soluble receptor 1, thrombospondin-1, microparticles, and microparticle-dependent procoagulant activity were measured in blood before treatment. The primary objective was 6-month progression-free survival (PFS). Twenty-seven evaluable patients were enrolled. Six-month PFS was 20.8%. Median PFS was 11.6 weeks (95% confidence interval: 7.5-15.7). Stable disease was the best response for nine (37.5%) patients, with a median duration of 11.2 weeks (4.2-35.85 weeks). No differences in PFS or response were observed among patients who relapsed during or after completion of adjuvant TMZ. Grade 3/4 adverse events included lymphopenia (15%), fatigue, diarrhea and febrile neutropenia (3.7% each), lymphopenia, neutropenia, and nausea/vomiting (11.1% each). One patient died from pneumonia and one patient died from pulmonary thromboembolism. Pretreatment levels of angiogenesis biomarkers, microparticles, and microparticle-related procoagulant activity were elevated in patients compared with healthy volunteers. This regimen is feasible, but failed to improve the results obtained with other second-line therapies in recurrent GB.

High correlation between 2 flow cytometry platforms in the microparticles analysis using a new calibrated beads strategy.

Microparticles (MPs) are potential noninvasive biomarkers for diagnosis or prognosis in pathologic conditions. However, the lack of standardization of the preanalytical and analytical methods leads to a wide variability in MPs results. The recently developed Megamix-Plus beads, a new bead-based standardization tool optimized to specific types of flow cytometers, could help circumvent this problem. The aim of the present study was to determine whether the number of total MPs and platelet-derived MPs (PMPs) is similar using 2 different cytometer platforms calibrated with the Megamix-Plus beads. Blood samples from 65 patients with deep venous thrombosis were collected and processed to obtain platelet poor plasma (PPP). The number of total MPs and PMPs in each PPP sample was measured using 2 flow cytometers. Megamix-Plus side scatter channel beads were used to calibrate the LSRFortessa flow cytometer from Becton Dickinson, whereas Megamix-Plus forward scatter channel beads were applied to the Navios flow cytometer from Beckman Coulter. High correlation of total MPs and PMPs values between the flow cytometers was found (r = 0.908, P < 0.01 and r = 0.910, P < 0.001, respectively). However, the absolute numbers of total MPs and PMPs were significantly higher measured with the Navios flow cytometer compared with the LSRFortessa cytometer. Therefore, both platforms are valid for MPs determination in general, although a similar platform with the same calibration tool could be a better choice for multicenter studies.

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 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 99(th) 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.

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 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.

Inflamación y disfunción endotelial en la insuficiencia cardíaca aguda y crónica / Endothelial dysfunction and inflammation in chronic and acute heart failure / Inflamação e disfunção endotelial na insuficiência cardíaca aguda e crônica

Introducción. En la insuficiencia cardíaca (IC) existe una importante activación neurohormonal e inflamatoria. También parece existir una disfunción endotelial. Nuestro objetivo ha sido comparar ambos procesos (inflamación y disfunción endotelial) en pacientes con IC. Material y métodos. Comparamos marcadores de disfunción endotelial (células endoteliales circulantes, macropartículas circulantes y factor von Willebrand) y de inflamación (proteína C reactiva, interleuquina 6 y fibrinógeno funcional) en 16 pacientes con insuficiencia cardíaca aguda (ICA), 16 con insuficiencia cardíaca crónica (ICC) estable y 32 controles sanos. Resultados. El número de células endoteliales circulantes fue mayor en los pacientes con ICA que en el grupo ICC y que en el grupo control (115,10 ± 63,44 vs 19,67 ± 3,17 vs 11,71 ± 2,92 cel/mL). La cantidad de macropartículas circulantes fue mayor en el grupo ICA que en el ICC y en ambos grupos frente al grupo control (9.627 ± 4.986 vs 3.970 ± 3.452 vs 1.371 ± 739 p/µL). El factor von Willebrand fue mayor en ambos grupos IC que en el control (234,3 ± 45,31 vs 245,92 ± 117,89 vs 100,14 ± 20,7%). Los valores de proteína C reactiva fueron mayores en el grupo ICA que en el ICC y que en el control (20,29 ± 17,56 vs 7,65 ± 4,27 vs 1,44 ± 1,10 mg/dL). La interleuquina 6 se encontró más elevada en los pacientes con ICA respecto al resto de grupos y en los pacientes con ICC respecto a los sanos (9,73 ± 9,37 vs 1,69 ± 1,36 vs 1,01 ± 1,09 pg/mL). Referente al fibrinógeno funcional, sólo encontramos diferencias significativas entre el grupo ICA y el resto de grupos (350 ± 60,48 vs 264,08 ± 67,02 vs 254,29 ± 23,69 mg/dL). Conclusiones. De forma paralela a la inflamación ya conocida en la IC, se produce una disfunción endotelial que a su vez parece ser proporcional a la gravedad de la IC.

Background. There is a neurohormonal an inflammatory activation in heart failure. There is also an endothelial dysfunction. Our objective war to compare both processes (inflammation and endothelial dysfunction) in patients with heart failure. Material and method. We compared endothelial dysfunction markers (circulating endothelial cells, circulating microparticles and Von Willebrand factor) and inflammatory markers (C reactive protein, interleukin-6 and functional fibrinogen) in 16 patients with acute heart failure (AHF), 16 with stable chronic heart failure (SHF) and 32 healthy controls. Results. The number of circulating endothelial cells was greater in AHF patients than in SHF and controls (115.10± 63.44 vs 19.67 ± 3.17 vs 11.71 ± 2.92 cel/mL). The amount of circulating microparticles was greater in the AHF group than in the SHF and in both than controls (9,627 ± 4,986 vs 3,970 ± 3,452 vs 1,371 ± 739 p/µL). Von Willebrand factor was greater in both heart failure groups than in controls (234.3 ± 45.31 vs 245.92 ± 117.89 vs 100.14± 20.7%). C reactive protein was greater in the AHF group than in the SHF group or controls (20.29 ± 17.56 vs 7.65± 4.27 vs 1.44 ± 1.10 mg/dL). Interleukin-6 was also higher in the AHF group than in the SHF and in this greater than in controls (9.73 ± 9.37 vs 1.69 ± 1.36 vs 1.01 ± 1.09 pg/mL). Functional fibrinogen was only greater in the AHF group (350 ± 60.48 vs 264.08 ± 67.02 vs 254.29 ± 23.69 mg/dL). Conclusions. Inflammation and endothelial dysfunction run together in heart failure patients. The endothelial dysfunction observed seems to be proportional to the inflammatory state.

Introdução. Na insuficiência cardíaca existe uma importante ativação neurohormonal e inflamatória. Também parece existir uma disfunção endotelial. Nosso objetivo foi o de comparar ambos processos (inflamação e disfunção endotelial) em pacientes com insuficiência cardíaca. Materiais e métodos. Comparamos marcadores de disfunção endotelial (células endoteliais circulantes, micropartículas circulantes e fator Von Willebrand) e de inflamação (proteína C reativa, interleuquina 6 e fibrinogênio funcional) em 16 pacientes com insuficiência cardíaca aguda (ICA), 16 com insuficiência cardíaca crônica estável (ICC) e 32 controles sãos. Resultados. O número de células endoteliais circulantes foi maior nos pacientes com ICA que no de ICC e que no controle (115,10 ± 63,44 vs 19,67 ± 3,17 vs 11,71 ± 2,92 cel/mL). A quantidade de micropartículas circulantes foi maior no grupo de ICA que no de ICC e em ambos grupos de pacientes em frente ao grupo controle (9.627 ± 4.986 vs 3.970 ± 3.452 vs 1.371 ± 739 p/µL). O fator Von Willebrand foi maior em ambos grupos de insuficiência cardíaca que no controle (234,3 ± 45,31 vs 245,92 ± 117,89 vs 100,14 ± 20,7%). Os valores de proteína C reativa foram maiores no grupo de ICA que no de ICC e que no de sãos (20,29 ± 17,56 vs 7,65 ± 4,27 vs 1,44 ± 1,10 mg/dL). A interleuquina seis encontrou-se mais elevada nos pacientes com ICA com respeito ao resto de grupos e nos pacientes com ICC com respeito aos sãos (9,73 ± 9,37 vs 1,69 ± 1,36 vs 1,01 ± 1,09 pg/mL). Com respeito ao fibrinogênio funcional só encontramos diferenças significativas entre o grupo de ICA e o resto de grupos (350 ± 60,48 vs 264,08± 67,02 vs 254,29 ± 23,69 mg/dL). Conclusões. De forma paralela à inflamação já conhecida na insuficiência cardíaca, se produz uma disfunção endotelial que a sua vez parece ser proporcional à gravidade da insuficiência cardíaca.

Inflamación y disfunción endotelial en la insuficiencia cardíaca aguda y crónica / Endothelial dysfunction and inflammation in chronic and acute heart failure / InflamaþÒo e disfunþÒo endotelial na insuficiÛncia cardíaca aguda e cr¶nica

Introducción. En la insuficiencia cardíaca (IC) existe una importante activación neurohormonal e inflamatoria. También parece existir una disfunción endotelial. Nuestro objetivo ha sido comparar ambos procesos (inflamación y disfunción endotelial) en pacientes con IC. Material y métodos. Comparamos marcadores de disfunción endotelial (células endoteliales circulantes, macropartículas circulantes y factor von Willebrand) y de inflamación (proteína C reactiva, interleuquina 6 y fibrinógeno funcional) en 16 pacientes con insuficiencia cardíaca aguda (ICA), 16 con insuficiencia cardíaca crónica (ICC) estable y 32 controles sanos. Resultados. El número de células endoteliales circulantes fue mayor en los pacientes con ICA que en el grupo ICC y que en el grupo control (115,10 ± 63,44 vs 19,67 ± 3,17 vs 11,71 ± 2,92 cel/mL). La cantidad de macropartículas circulantes fue mayor en el grupo ICA que en el ICC y en ambos grupos frente al grupo control (9.627 ± 4.986 vs 3.970 ± 3.452 vs 1.371 ± 739 p/AL). El factor von Willebrand fue mayor en ambos grupos IC que en el control (234,3 ± 45,31 vs 245,92 ± 117,89 vs 100,14 ± 20,7%). Los valores de proteína C reactiva fueron mayores en el grupo ICA que en el ICC y que en el control (20,29 ± 17,56 vs 7,65 ± 4,27 vs 1,44 ± 1,10 mg/dL). La interleuquina 6 se encontró más elevada en los pacientes con ICA respecto al resto de grupos y en los pacientes con ICC respecto a los sanos (9,73 ± 9,37 vs 1,69 ± 1,36 vs 1,01 ± 1,09 pg/mL). Referente al fibrinógeno funcional, sólo encontramos diferencias significativas entre el grupo ICA y el resto de grupos (350 ± 60,48 vs 264,08 ± 67,02 vs 254,29 ± 23,69 mg/dL). Conclusiones. De forma paralela a la inflamación ya conocida en la IC, se produce una disfunción endotelial que a su vez parece ser proporcional a la gravedad de la IC.(AU)

Background. There is a neurohormonal an inflammatory activation in heart failure. There is also an endothelial dysfunction. Our objective war to compare both processes (inflammation and endothelial dysfunction) in patients with heart failure. Material and method. We compared endothelial dysfunction markers (circulating endothelial cells, circulating microparticles and Von Willebrand factor) and inflammatory markers (C reactive protein, interleukin-6 and functional fibrinogen) in 16 patients with acute heart failure (AHF), 16 with stable chronic heart failure (SHF) and 32 healthy controls. Results. The number of circulating endothelial cells was greater in AHF patients than in SHF and controls (115.10± 63.44 vs 19.67 ± 3.17 vs 11.71 ± 2.92 cel/mL). The amount of circulating microparticles was greater in the AHF group than in the SHF and in both than controls (9,627 ± 4,986 vs 3,970 ± 3,452 vs 1,371 ± 739 p/AL). Von Willebrand factor was greater in both heart failure groups than in controls (234.3 ± 45.31 vs 245.92 ± 117.89 vs 100.14± 20.7%). C reactive protein was greater in the AHF group than in the SHF group or controls (20.29 ± 17.56 vs 7.65± 4.27 vs 1.44 ± 1.10 mg/dL). Interleukin-6 was also higher in the AHF group than in the SHF and in this greater than in controls (9.73 ± 9.37 vs 1.69 ± 1.36 vs 1.01 ± 1.09 pg/mL). Functional fibrinogen was only greater in the AHF group (350 ± 60.48 vs 264.08 ± 67.02 vs 254.29 ± 23.69 mg/dL). Conclusions. Inflammation and endothelial dysfunction run together in heart failure patients. The endothelial dysfunction observed seems to be proportional to the inflammatory state.(AU)

IntroduþÒo. Na insuficiÛncia cardíaca existe uma importante ativaþÒo neurohormonal e inflamatória. Também parece existir uma disfunþÒo endotelial. Nosso objetivo foi o de comparar ambos processos (inflamaþÒo e disfunþÒo endotelial) em pacientes com insuficiÛncia cardíaca. Materiais e métodos. Comparamos marcadores de disfunþÒo endotelial (células endoteliais circulantes, micropartículas circulantes e fator Von Willebrand) e de inflamaþÒo (proteína C reativa, interleuquina 6 e fibrinogÛnio funcional) em 16 pacientes com insuficiÛncia cardíaca aguda (ICA), 16 com insuficiÛncia cardíaca cr¶nica estável (ICC) e 32 controles sÒos. Resultados. O número de células endoteliais circulantes foi maior nos pacientes com ICA que no de ICC e que no controle (115,10 ± 63,44 vs 19,67 ± 3,17 vs 11,71 ± 2,92 cel/mL). A quantidade de micropartículas circulantes foi maior no grupo de ICA que no de ICC e em ambos grupos de pacientes em frente ao grupo controle (9.627 ± 4.986 vs 3.970 ± 3.452 vs 1.371 ± 739 p/AL). O fator Von Willebrand foi maior em ambos grupos de insuficiÛncia cardíaca que no controle (234,3 ± 45,31 vs 245,92 ± 117,89 vs 100,14 ± 20,7%). Os valores de proteína C reativa foram maiores no grupo de ICA que no de ICC e que no de sÒos (20,29 ± 17,56 vs 7,65 ± 4,27 vs 1,44 ± 1,10 mg/dL). A interleuquina seis encontrou-se mais elevada nos pacientes com ICA com respeito ao resto de grupos e nos pacientes com ICC com respeito aos sÒos (9,73 ± 9,37 vs 1,69 ± 1,36 vs 1,01 ± 1,09 pg/mL). Com respeito ao fibrinogÛnio funcional só encontramos diferenþas significativas entre o grupo de ICA e o resto de grupos (350 ± 60,48 vs 264,08± 67,02 vs 254,29 ± 23,69 mg/dL). Conclus§es. De forma paralela O inflamaþÒo já conhecida na insuficiÛncia cardíaca, se produz uma disfunþÒo endotelial que a sua vez parece ser proporcional O gravidade da insuficiÛncia cardíaca.(AU)

Inflamación y disfunción endotelial en la insuficiencia cardíaca aguda y crónica / Endothelial dysfunction and inflammation in chronic and acute heart failure / Inflamação e disfunção endotelial na insuficiência cardíaca aguda e crônica

Introducción. En la insuficiencia cardíaca (IC) existe una importante activación neurohormonal e inflamatoria. También parece existir una disfunción endotelial. Nuestro objetivo ha sido comparar ambos procesos (inflamación y disfunción endotelial) en pacientes con IC. Material y métodos. Comparamos marcadores de disfunción endotelial (células endoteliales circulantes, macropartículas circulantes y factor von Willebrand) y de inflamación (proteína C reactiva, interleuquina 6 y fibrinógeno funcional) en 16 pacientes con insuficiencia cardíaca aguda (ICA), 16 con insuficiencia cardíaca crónica (ICC) estable y 32 controles sanos. Resultados. El número de células endoteliales circulantes fue mayor en los pacientes con ICA que en el grupo ICC y que en el grupo control (115,10 ñ 63,44 vs 19,67 ñ 3,17 vs 11,71 ñ 2,92 cel/mL). La cantidad de macropartículas circulantes fue mayor en el grupo ICA que en el ICC y en ambos grupos frente al grupo control (9.627 ñ 4.986 vs 3.970 ñ 3.452 vs 1.371 ñ 739 p/µL). El factor von Willebrand fue mayor en ambos grupos IC que en el control (234,3 ñ 45,31 vs 245,92 ñ 117,89 vs 100,14 ñ 20,7%). Los valores de proteína C reactiva fueron mayores en el grupo ICA que en el ICC y que en el control (20,29 ñ 17,56 vs 7,65 ñ 4,27 vs 1,44 ñ 1,10 mg/dL). La interleuquina 6 se encontró más elevada en los pacientes con ICA respecto al resto de grupos y en los pacientes con ICC respecto a los sanos (9,73 ñ 9,37 vs 1,69 ñ 1,36 vs 1,01 ñ 1,09 pg/mL). Referente al fibrinógeno funcional, sólo encontramos diferencias significativas entre el grupo ICA y el resto de grupos (350 ñ 60,48 vs 264,08 ñ 67,02 vs 254,29 ñ 23,69 mg/dL). Conclusiones. De forma paralela a la inflamación ya conocida en la IC, se produce una disfunción endotelial que a su vez parece ser proporcional a la gravedad de la IC.(AU)

Background. There is a neurohormonal an inflammatory activation in heart failure. There is also an endothelial dysfunction. Our objective war to compare both processes (inflammation and endothelial dysfunction) in patients with heart failure. Material and method. We compared endothelial dysfunction markers (circulating endothelial cells, circulating microparticles and Von Willebrand factor) and inflammatory markers (C reactive protein, interleukin-6 and functional fibrinogen) in 16 patients with acute heart failure (AHF), 16 with stable chronic heart failure (SHF) and 32 healthy controls. Results. The number of circulating endothelial cells was greater in AHF patients than in SHF and controls (115.10ñ 63.44 vs 19.67 ñ 3.17 vs 11.71 ñ 2.92 cel/mL). The amount of circulating microparticles was greater in the AHF group than in the SHF and in both than controls (9,627 ñ 4,986 vs 3,970 ñ 3,452 vs 1,371 ñ 739 p/µL). Von Willebrand factor was greater in both heart failure groups than in controls (234.3 ñ 45.31 vs 245.92 ñ 117.89 vs 100.14ñ 20.7%). C reactive protein was greater in the AHF group than in the SHF group or controls (20.29 ñ 17.56 vs 7.65ñ 4.27 vs 1.44 ñ 1.10 mg/dL). Interleukin-6 was also higher in the AHF group than in the SHF and in this greater than in controls (9.73 ñ 9.37 vs 1.69 ñ 1.36 vs 1.01 ñ 1.09 pg/mL). Functional fibrinogen was only greater in the AHF group (350 ñ 60.48 vs 264.08 ñ 67.02 vs 254.29 ñ 23.69 mg/dL). Conclusions. Inflammation and endothelial dysfunction run together in heart failure patients. The endothelial dysfunction observed seems to be proportional to the inflammatory state.(AU)

Introdução. Na insuficiência cardíaca existe uma importante ativação neurohormonal e inflamatória. Também parece existir uma disfunção endotelial. Nosso objetivo foi o de comparar ambos processos (inflamação e disfunção endotelial) em pacientes com insuficiência cardíaca. Materiais e métodos. Comparamos marcadores de disfunção endotelial (células endoteliais circulantes, micropartículas circulantes e fator Von Willebrand) e de inflamação (proteína C reativa, interleuquina 6 e fibrinogênio funcional) em 16 pacientes com insuficiência cardíaca aguda (ICA), 16 com insuficiência cardíaca crônica estável (ICC) e 32 controles sãos. Resultados. O número de células endoteliais circulantes foi maior nos pacientes com ICA que no de ICC e que no controle (115,10 ñ 63,44 vs 19,67 ñ 3,17 vs 11,71 ñ 2,92 cel/mL). A quantidade de micropartículas circulantes foi maior no grupo de ICA que no de ICC e em ambos grupos de pacientes em frente ao grupo controle (9.627 ñ 4.986 vs 3.970 ñ 3.452 vs 1.371 ñ 739 p/µL). O fator Von Willebrand foi maior em ambos grupos de insuficiência cardíaca que no controle (234,3 ñ 45,31 vs 245,92 ñ 117,89 vs 100,14 ñ 20,7%). Os valores de proteína C reativa foram maiores no grupo de ICA que no de ICC e que no de sãos (20,29 ñ 17,56 vs 7,65 ñ 4,27 vs 1,44 ñ 1,10 mg/dL). A interleuquina seis encontrou-se mais elevada nos pacientes com ICA com respeito ao resto de grupos e nos pacientes com ICC com respeito aos sãos (9,73 ñ 9,37 vs 1,69 ñ 1,36 vs 1,01 ñ 1,09 pg/mL). Com respeito ao fibrinogênio funcional só encontramos diferenças significativas entre o grupo de ICA e o resto de grupos (350 ñ 60,48 vs 264,08ñ 67,02 vs 254,29 ñ 23,69 mg/dL). Conclusões. De forma paralela à inflamação já conhecida na insuficiência cardíaca, se produz uma disfunção endotelial que a sua vez parece ser proporcional à gravidade da insuficiência cardíaca.(AU)

Are there differences in acute phase inflammation markers regarding the type of heart failure?

This study aimed to determine if there are differences in inflammatory markers in the acute phase between systolic heart failure and heart failure with preserved systolic function. One hundred and thirty-one patients with acute heart failure were recruited consecutively. At admission, plasma fibrinogen, C-reactive protein, sialic acid, von Willebrand factor, vascular endothelial growth factor, interleukin-6 and NTproBNP were all evaluated. If the ejection fraction was 45% or over patients were included in the HF-PSF group; the remaining patients were included in the SHF group. The HF-PSF patients were older (72±10 vs 63±12 years, P<0.001), presented a higher rate of atrial fibrillation (56.1 vs 21.3%, P<0.001), and had a lower rate of hemoglobin (12.2±2 vs 13.3±2.1 g/dL, P<0.01). No significant differences were observed in the inflammation markers analyzed among SHF and HF-PSF groups. In the acute phase of heart failure there is a marked elevation of inflammatory markers but there are no differences in the inflammatory markers analyzed between the two different types of heart failure.

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.

Células endoteliales y micropartículas circulantes en pacientes portadores de anticuerpos antifosfolípidos / Circulating endothelial cells and microparticles in patients with antiphospholipid antibodies

Fundamento y objetivo: Determinar si los valores de células endoteliales circulantes (CEC), micropartículas circulantes (MP) y factor von Willebrand (FvW), marcadores establecidos de disfunción/daño endotelial, están elevados en pacientes portadores de anticuerpos antifosfolípidos (AAF) y si existe correlación con marcadores de inflamación y coagulación. Pacientes y método: Se estudian 12 pacientes portadores de AAF y 12 sujetos sanos. Se determinaron CEC, MP, FvW, proteína C reactiva (PCR), fibrinógeno (Fg), ácido siálico (AS), interleucina 6, factor tisular (FT), generación de trombina (GT) y fragmentos de protrombina (F1+2) y de fibrina (DD). Resultados: En los pacientes están significativamente elevados los valores de los marcadores de: disfunción/daño endotelial (CEC, MP y FvW), inflamación (Fg y PCR) y coagulación (FT y DD). El análisis bivariante muestra correlación significativa entre CEC y Fg, AS, PCR y DD, así como entre CEC y FvW y MP. Conclusión: Los pacientes portadores de AAF presentan una disfunción endotelial asociada a un proceso inflamatorio, que, unido a los valores elevados de Fg, FT y DD, puede inducir un estado de hipercoagulabilidad (AU)

Background and objective: To determine whether circulating endothelial cells (CECs), circulatingmicroparticles (MPs) and von Willebrand factor (vWF), established markers of endothelial dysfunction/ damage, are elevated in patients with antiphospholipid antibodies (aPL) and its possible correlation with inflammation and coagulation. Patients and methods: Twelve atients with aPL and 12 healthy subjects were studied. Levels of CECs, MPs, vWF, C reactive protein (CRP), fibrinogen (Fg), sialic acid (SA), interleukin 6 (IL-6), tissue factor (TF), thrombin generation (TG) and prothrombin (F1 + 2) and fibrin (DD) fragments were determined. Results: In patients, markers of dysfunction/damage endothelial, CECs, MPs and vWF; inflammation, Fgand CRP and coagulation, TF and DD were significantly elevated. The bivariate analysis showedsignificant correlation among CECs and Fg, AS, CRP and DD, as well as between CECs and vWF and MPs.Conclusion: Patients with aPL had endothelial dysfunction associated with an inflammatory process,which, together with high levels of Fg, TF and DD, may be responsible for the hypercoagulable state (AU)

[Circulating endothelial cells and microparticles in patients with antiphospholipid antibodies]. / Células endoteliales y micropartículas circulantes en pacientes portadores de anticuerpos antifosfolípidos.

BACKGROUND AND OBJECTIVE: To determine whether circulating endothelial cells (CECs), circulating microparticles (MPs) and von Willebrand factor (vWF), established markers of endothelial dysfunction/damage, are elevated in patients with antiphospholipid antibodies (aPL) and its possible correlation with inflammation and coagulation. PATIENTS AND METHODS: Twelve patients with aPL and 12 healthy subjects were studied. Levels of CECs, MPs, vWF, C reactive protein (CRP), fibrinogen (Fg), sialic acid (SA), interleukin 6 (IL-6), tissue factor (TF), thrombin generation (TG) and prothrombin (F1+2) and fibrin (DD) fragments were determined. RESULTS: In patients, markers of dysfunction/damage endothelial, CECs, MPs and vWF; inflammation, Fg and CRP and coagulation, TF and DD were significantly elevated. The bivariate analysis showed significant correlation among CECs and Fg, AS, CRP and DD, as well as between CECs and vWF and MPs. CONCLUSION: Patients with aPL had endothelial dysfunction associated with an inflammatory process, which, together with high levels of Fg, TF and DD, may be responsible for the hypercoagulable state.

Atorvastatin neutralises the thrombin-induced tissue factor expresion in endothelial cells via geranylgeranyl pyrophosphate.

Statins may have beneficial effects in atherogenesis given their antithrombotic properties involving non-lipid mechanisms that modify endothelial function of tissue factor induction by thrombin. In this study, we investigate the effect of atorvastatin on tissue factor (TF) activity in thrombin-stimulated endothelial cells and its regulation through mevalonate or its derivatives. First subculture of human umbilical endothelial cells was used for this study. Cells were treated with thrombin and atorvastatin for different time intervals and dosage. Tissue factor activity was measured as Factor Xa generation induced by Tissue Factor-Factor VIIa complex on confluent cells. Our results show that atorvastatin prevents the thrombin-induced up-regulation of tissue factor activity in a concentration-dependent manner. Mevalonate and geranylgeranyl pyrophosphate reversed this inhibitory effect of atorvastatin on tissue factor activity, while the presence of farnesyl pyrophosphate did not prevent the atorvastatin effect on thrombin-induced tissue factor activity. Rho-kinase inhibitor did not affect the thrombin stimulation of tissue factor activity. High amount of hydrophobic isoprenoid groups decreases the thrombin-induced TF activity and may promote endothelial cell anti-thrombotic action. Rho kinase pathways do not have a major role in the thrombin-mediated TF activity. The inhibitory effect of atorvastatin on thrombin-induced TF activity was partially reversed by MVA and GGPP but not FPP.

Circulating markers of angiogenesis, inflammation, and coagulation in patients with glioblastoma.

Inflammation, angiogenesis, and coagulation are linked to the development of cancer. In glioblastoma, microvascular proliferation is a hallmark, and lymphocytic infiltration is a common finding. Thromboses are frequent in patients with glioblastoma. The objective of this study was to assess presurgical levels of circulating markers of inflammation, angiogenesis, and coagulation in a prospective series of patients with glioblastoma, and to explore their correlations and possible associations with clinical findings. Angiogenesis markers included were vascular endothelial growth factor (VEGF), soluble vascular endothelial growth factor-receptor 1 (sVEGFR-1), and thrombospondin-1 (TSP-1). Inflammatory markers included were C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and sialic acid (SA). Coagulation markers included were fibrinogen (Fg), endogen thrombin generation (ETG), prothrombin fragments 1 + 2 (F1 + 2), and tissue factor (TF). Forty-seven patients and 60 healthy subjects were included in the study. Signs of tumor necrosis in presurgical MRI were associated with shorter survival (P < 0.01). All inflammation markers, F1 + 2, ETG, VEGF and sVEGFR-1, were significantly elevated in glioblastoma patients. Correlations were found between ETG and Fg (r = 0.44, P < 0.01). Sialic acid correlated with Fg (r = 0.63, P < 0,001); CPR correlated with SA (r = 0.60, P < 0.001), Fg (r = 0.76, P < 0.001), TNFα (r = 0.56, P < 0.001), and IL-6 (r = 0.65, P < 0.001); and IL-6 also correlated positively with TNFα (r = 0.40, P < 0.02) and Fg (r = 0.45, P < 0.01). Vascular endothelial growth factor inversely correlated with sVEGFR-1 (r = -0.35, P < 0.02). No associations were found between marker levels and survival or progression-free survival.

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.

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.

Inflammatory markers in stable heart failure and their relationship with functional class.

INTRODUCTION AND OBJECTIVES: While it appears to be clear that an inflammatory process occurs in heart failure (HF), it is still to be defined whether inflammation depends to a greater extent on HF etiology, functional class (FC), or the extent of depression of ejection fraction (EF). Our objectives were to analyze differences in inflammatory marker levels as compared to a healthy population, to assess differences depending on HF etiology, and to relate values with FC and EF. PATIENTS AND METHODS: Fifty-nine consecutive outpatients with stable HF (57 + or - 9 years, 89% males) and 59 controls (55 + or - 8 years, 85% males) were enrolled into the study. Causes of HF included ischemic heart disease (n=24), idiopathic dilated cardiomyopathy (n=24), and miscellaneous conditions (n=11). Patients with decompensation in the past 6 months were excluded from the study. Protein fibrinogen, sialic acid, C-reactive protein (CRP), and tumor necrosis factor-alpha (TNF-alpha) were measured. Echocardiography was performed in all study patients. FC was assessed using the NYHA classification. RESULTS: A comparison of inflammatory marker levels between the HF and control groups showed significant differences in all markers, except for TNF-alpha. Protein fibrinogen in controls: 253 + or - 54 mg/dl, protein fibrinogen in HF: 294 + or - 67 mg/dl; p<0.05. Sialic acid in controls: 53 + or - 1 mg/dl, sialic acid in HF: 61 + or - 12 mg/dl; p<0.05. CRP in controls: 1.3 + or - 0.7 mg/dl, CRP in HF: 7.8 + or - 1.2 mg/dl; p<0.05. TNF-alpha in controls: 183 + or - 51 ng/ml, TNF-alpha in HF: 203 + or - 13 ng/ml; p=0.2. No differences were found between the different etiologies of HF. A positive association was seen between FC and protein fibrinogen and TNF-alpha (p<0.05), but not with EF. CONCLUSIONS: Increased inflammatory marker levels related to FC of the patient, but not to EF, are found in chronic HF.

Inflammation, endothelial dysfunction and angiogenesis markers in chronic heart failure patients.

This study was conducted to assess the relationship among circulating markers of inflammation, endothelial dysfunction and angiogenesis in 59 chronic heart failure (CHF) patients. Increased concentrations of C-reactive protein (CRP), tumour necrosis factor-alpha (TNF-alpha), von Willebrand factor (VWF) and fibrinogen are strongly implicated in the development of CHF. Increased vascular endothelium grow factor (VEGF) and decreased thrombospondin-1 (TSP-1) concentrations suggest a role of angiogenesis in the maintenance and repair of luminal endothelium in CHF. A relationship among markers of endothelial dysfunction (VWF) and inflammation (fibrinogen, CRP) and angiogenesis (VEGF, TSP-1) was found in CHF patients.