[show abstract][hide abstract] ABSTRACT: Circulating endothelial cells (CECs) are a novel and valuable marker of endothelial damage in a variety of vascular disorders. There is limited information as to CEC counts and the time course of CECs in subtypes of stroke.
We studied 49 patients with stroke (18 with atherothrombotic infarction in the territory of the middle cerebral artery, 16 with cardioembolic stroke, and 15 with lacunar stroke). We also included 16 healthy controls and 64 disease controls. CECs were isolated and enumerated with lectin-augmented CD146-driven immunomagnetic isolation. Neurologic deficit was assessed with the European Stroke Scale (ESS) and the National Institutes of Health Stroke Scale (NIHSS). Recovery was assessed with the modified Rankin scale (mRS).
Healthy controls had low numbers of CECs (median, 8 cells/mL; mean, 9 cells/mL; range, 0-16 cells/mL; n = 16). Patients with stroke had markedly elevated numbers of CECs at presentation. Patients with atherothrombotic infarction had 32 cells per milliliter (mean, 42 cells/mL; range, 24-116 cells/mL; n = 18; P < .001 when compared to controls). Patients with lacunar stroke had 68 cells per milliliter (mean, 68 cells/mL; range, 8-144 cells/mL; n = 15; P < .001 when compared to controls). Patients with cardioembolic stroke had 46 cells per milliter (mean, 54 cells/mL; range, 24-116 cells/mL; n = 16; P < .001 when compared to healthy controls). There was a tendency towards higher numbers of CECs in lacunar stroke. The number of CECs peaked at day 7 in patients with atherothrombotic infarction and came back to normal at day 90. In contrast, CECs in patients with acute lacunar stroke and cardioembolic stroke decreased progressively until day 90.
CECs are markers of endothelial damage and/or repair in stroke. Differences during the course of disease are likely to reflect different pathophysiology.
Journal of stroke and cerebrovascular diseases: the official journal of National Stroke Association 08/2011; 21(6):452-8.
[show abstract][hide abstract] ABSTRACT: Higher rates of acute rejection (AR) and reduced graft survival have been reported in patients with cytomegalovirus (CMV) infection, but an association between these factors remains controversial.
In this study, serial protocol biopsies (PBs) and clinically indicated biopsies (IBs) from a large cohort of renal allograft recipients (n ¼ 594) were analyzed to examine the relation between CMV and AR.
Patients with CMV were more likely to receive IB (85 of the 153 patients; 56%) compared to patients without CMV (138 of 441 patients; 32%; P = 0.003). However, this did not translate into a greater number of patients with episodes of acute cellular rejection on histopathology in IBs. Analysis of PBs revealed a significantly higher number of episodes of rejection per patient with CMV infection (P = 0.04), but only in a subgroup of patients with triple immunosuppression. Long-term graft function post-transplantation was analyzed in four different subgroups according to CMV infection and/or AR. Differences in renal function were apparent within the first 6 weeks after transplantation and persisted during follow-up, with the best renal function in patients without AR or CMV, whereas patients with both AR and CMV had the worst (P < 0.012 at 1 year; P < 0.001 at 2 years). On average, the latter group had significantly older donors and more often delayed graft function.
Our data suggests that the link between CMV and AR is far less significant than previously thought. Outcome in patients with CMV may be more determined by coexisting conditions like high donor age and delayed graft function.
[show abstract][hide abstract] ABSTRACT: About 30 years ago circulating endothelial cells (CEC) were first observed in peripheral blood. Since then CEC have been established as a reliable indicator of vascular injury and damage and more sophisticated detection techniques, such as immunomagnetic isolation and fluorescence-activated cell sorting (FACS), have become available. However even today there remains controversy as to the best approach to isolate and enumerate these cells. Here, we review the isolation and enumeration of CEC with an emphasis on CD146-driven immunomagnetic isolation and FACS as the two competing techniques. We describe advantages and pitfalls of both approaches. Moreover, we provide a list of clinical studies in this field and describe the possible clinical utility of CEC as a surrogate marker for vascular damage and dysfunction. In addition, we review the phenotype of CEC and discuss mechanisms of detachment. Recent evidence has also revealed interesting interactions between CEC and healthy endothelium in vitro although the relevance of these findings for human vascular disease in vivo remains unclear. Finally, we highlight differences between circulating endothelial cells and endothelial progenitor cells. In summary, CEC must be regarded as a sensitive and specific marker of endothelial damage as well as a potential mediator in vascular disease.
Current Stem Cell Research & Therapy 12/2010; 5(4):294-302. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: The clinical importance of microparticles resulting from vesiculation of platelets and other blood cells is increasingly recognized, although no standardized method exists for their measurement. Only a few studies have examined the analytical and preanalytical steps and variables affecting microparticle detection. We focused our analysis on microparticle detection by flow cytometry. The goal of our study was to analyze the effects of different centrifugation protocols looking at different durations of high and low centrifugation speeds. We also analyzed the effect of filtration of buffer and long-term freezing on microparticle quantification, as well as the role of Annexin V in the detection of microparticles. Absolute and platelet-derived microparticles were 10- to 15-fold higher using initial lower centrifugation speeds at 1500 × g compared with protocols using centrifugation speeds at 5000 × g (P < 0.01). A clear separation between true events and background noise was only achieved using higher centrifugation speeds. Filtration of buffer with a 0.2 μm filter reduced a significant amount of background noise. Storing samples for microparticle detection at -80°C decreased microparticle levels at days 28, 42, and 56 (P < 0.05 for all comparisons with fresh samples). We believe that staining with Annexin V is necessary to distinguish true events from cell debris or precipitates. Buffers should be filtered and fresh samples should be analyzed, or storage periods will have to be standardized. Higher centrifugation speeds should be used to minimize contamination by smaller size platelets.
Vascular Health and Risk Management 01/2010; 6:1125-33.
[show abstract][hide abstract] ABSTRACT: Much has been learned in recent years on the pathogenesis of ANCA-associated small-vessel vasculitis. The interaction of primed neutrophils with ANCA and endothelial cells is crucial to the disease. Next we gained a better understanding, from animal models, of the pathogenetic importance of the ANCA antibody. Very recent evidence provides intriguing data regarding the link between infection and vasculitis, LAMP-2 antibodies as novel markers, and NETs as a novel pathogenetic mechanism. It remains to be seen whether others are able to corroborate these findings and whether testing for LAMP-2 antibodies will become part of the clinical routine in vasculitis. Recent years also saw the emergence of various new markers of endothelial damage and the disease itself, such as circulating endothelial cells and endothelial microparticles. These novel markers correlate well with disease activity; they may well complement traditional diagnostic tools, such as ANCA testing. Preliminary evidence has provided some insight into the balance between endothelial damage and repair. Exciting preliminary data also indicate that circulating endothelial cells may not only be markers of disease activity but that these cells may have pathogenetic importance in their own right. These findings may have profound implications for the pathogenesis of vasculitis and vascular disease in general. Recent years also saw the publication of a number of seminal studies for the treatment of ANCA-associated vasculitis. We now have a much better understanding of the role of pulse intravenous cyclophosphamide and plasma exchange than ten or even five years ago. Further studies must now show whether plasma exchange is also beneficial for less severely ill patients with AASV. Finally, as ever, it is hoped that further progress in understanding the disease pathogenesis will also provide more tailored and less toxic therapies.
Minerva urologica e nefrologica = The Italian journal of urology and nephrology 12/2009; 61(4):411-37. · 0.63 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rapid apoptotic cell engulfment is crucial for prevention of inflammation and autoimmune diseases and is conducted by special immunocompetent cells like macrophages or immature dendritic cells. We recently demonstrated that endothelial cells (ECs) also participate in apoptotic cell clearance. However, in contrast to conventional phagocytes they respond with an inflammatory phenotype. To further confirm these pro-inflammatory responses human ECs were exposed to apoptotic murine ECs and changes in thrombospondin-1 (TSP-1) expression and in activation of intracellular signalling cascades were determined by real-time qPCR, immunoblotting and immunocytochemistry. Human primary macrophages or monocytic lymphoma cells (U937) were incubated with conditioned supernatant of human ECs exposed to apoptotic cells and changes in activation, migration and phagocytosis were monitored. Finally, plasma levels of TSP-1 in patients with anti-neutrophil cytoplasmic antibody(ANCA)-associated vasculitis (AAV) were determined by ELISA. We provided evidence that apoptotic cells induce enhanced expression of TSP-1 in human ECs and that this increase in TSP-1 is mediated by the mitogen-activated protein kinases (MAPK) ERK1 and 2 and their upstream regulators MEK and B-Raf. We also showed that plasma TSP-1 levels are increased in patients with AAV. Finally, we showed that conditioned supernatant of ECs exposed to apoptotic cells induces pro-inflammatory responses in monocytes or U937 cells and demonstrated that increased TSP-1 expression enhances migration and facilitates engulfment of apoptotic cells by monocyte-derived macrophages or U937 cells. These findings suggest that under pathological conditions with high numbers of uncleared dying cells in the circulation endothelial-derived elevated TSP-1 level may serve as an attraction signal for phagocytes promoting enhanced recognition and clearance of apoptotic cells.
Journal of Cellular and Molecular Medicine 07/2009; 14(7):1922-34. · 4.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: Endothelial cells play a central pathogenetic role in ANCA-associated small-vessel vasculitis (AAV). Circulating endothelial cells (CECs), as a marker of endothelial damage, have been shown to be elevated in vasculitis. More recently, endothelial microparticles (EMPs) were found to be increased in active childhood vasculitis. The role of EMP in adult AAV and the relationship between EMP and CEC is unclear.
We studied 26 patients with AAV, 12 healthy volunteers and 10 patients with IgA nephropathy as disease control. Platelet-poor plasma was ultracentrifuged. MPs were identified and enumerated with flow cytometry, Annexin V, CD62E and CD105 antibodies. Leucocyte- and platelet-derived MPs were also measured. CEC were isolated and enumerated with CD146-driven immuno-magnetic isolation.
EMPs are significantly elevated in patients with active vasculitis (CD62E: mean 248 MP/microl +/- 198 s.d.; CD105: 121 +/- 135/microl) compared with patients in remission/partial remission (CD62E: 55 +/- 30/microl, P = 0.001; CD105: 16 +/- 12/microl, P = 0.002) and healthy volunteers (CD62E: 66 +/- 33/microl, P = 0.002; CD105: 25 +/- 26/microl, P = 0.007). The MP count correlates with disease activity measured by the Birmingham Vasculitis Activity Score (BVAS) (CD62E: r = 0.703; CD105: r = 0.445, P < 0.023).
EMPs are elevated in active adult AAV. EMP levels correlate with disease activity and might serve as a marker of endothelial activation and damage. Differential detection of endothelial, platelet- and leucocyte-derived MPs may provide more insight in to the pathogenesis of AAV.
[show abstract][hide abstract] ABSTRACT: B-cell depletion with rituximab, a chimeric anti-CD20 antibody, is a novel treatment for refractory and relapsing ANCA-associated small-vessel vasculitis. Data are limited and most reports describe single patients or small numbers of patients followed prospectively.
We report a single-centre experience with 15 patients who received rituximab for refractory or relapsing ANCA-associated vasculitis. All patients had been treated with corticosteroids and cyclophosphamide and a variety of other second-line immunosuppressive agents. None of the patients had evidence of infection and received four infusions of 375 mg/m(2) of rituximab. Disease activity was assessed in accordance with the Birmingham Vasculitis Activity Score (BVAS). BVAS, C-reactive protein and ANCA titres were recorded at baseline and during follow-up.
B-cell depletion was achieved in all patients. Partial or complete remission was seen in 14 of 15 patients with a significant decline in BVAS compared to baseline (P < 0.007). One patient with granulomatous ANCA-associated vasculitis did not respond to rituximab. There were no side effects during rituximab infusion. Transient leucopenia was observed in two patients. One patient with bronchial stenosis died of pneumonia 5.5 months after the initiation of rituximab treatment. One initially anti-HBc-positive/HBsAg-negative patient experienced a reactivation of hepatitis B, developed end-stage renal failure and died after refusal of dialysis.
We report the largest case series of rituximab use for ANCA-associated vasculitis so far. Our data support that the drug is capable of inducing partial or complete remission in refractory or relapsing patients. Leucopenia and infectious complications remain a matter of concern.
[show abstract][hide abstract] ABSTRACT: Acute tubular injury (ATI) is commonly observed in renal allografts, especially early after transplantation. This study analyzes prevalence and associated clinical conditions of ATI in serial protocol biopsies (pBx) and indication biopsies (iBx), and its impact on long-term graft function. 612 pBx from 204 patients taken at 6 weeks, 3 and 6 months, and 151 iBx performed within the first year of transplantation were evaluated. Prevalence of ATI in pBx was 40% (6 weeks), 34% (3 months) and 37% (6 months), and 46% in iBx. ATI was associated with delayed graft function and prolonged cold ischemia time in pBx, and with acute rejections in iBx. The GFR at 1 and 2 years after transplantation correlated inversely with the frequency of ATI in both pBx and iBx (p < 0.001). Prevalence of chronic changes at 6 months was not significantly related to ATI (patients without ATI: 36%, patients with multiple ATI findings: 54%). ATI is linked to inferior long-term graft function. While this suggests lack of recovery from ATI with permanent allograft damage, the underlying molecular mechanisms need yet to be uncovered. Prevention of the potential pathogenetic factors identified in this study might be the key point to attain good long-term graft function.
American Journal of Transplantation 06/2008; 8(8):1684-93. · 6.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Preeclampsia is a disorder of endothelial cells, and novel markers of the disease are eagerly awaited. We tested the hypothesis that circulating endothelial cells (CECs) are elevated in preeclampsia and that cell numbers correlate with disease activity.
CECs were measured in 10 patients with preeclampsia as well as pregnant and nonpregnant controls. Cells were enumerated prior to delivery, 1 and 3-5 days thereafter. Enumeration of CECs was performed with anti-CD 146-driven immunomagnetic isolation and subsequent Ulex lectin staining.
Markedly elevated CEC numbers were detected in women with preeclampsia (median 88 cells/mL; P < .001) when compared with normal pregnancies (median 16 cells/mL) and healthy nonpregnant women (12 cells/mL). There was a significant correlation of CEC numbers and systolic blood pressure (P < .02). A rapid decline of cell numbers after delivery paralleled the clinical recovery.
Circulating endothelial cells are a novel marker of vascular damage in preeclampsia.
American journal of obstetrics and gynecology 04/2008; 198(3):317.e1-5. · 3.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Circulating endothelial cells (CECs) have been detected in a variety of vascular disorders, but their interactions with healthy endothelium remain unknown. The aim of this study was to evaluate the response of human endothelial cells (ECs) to apoptotic or necrotic ECs in an in vitro model and to delineate pathogenetic pathways. Here we show that incubation of the human microvascular endothelial cell line (HMEC-1) with apoptotic ECs resulted in increased expression of chemokines and enhanced binding of leukocytes to HMEC-1 cells, whereas exposure of HMEC-1 cells to necrotic ECs caused no changes in leukocyte-binding affinity. Both apoptotic and necrotic cells were bound and engulfed by HMEC-1 cells and primary human umbilical vein endothelial cells (HUVECs). We therefore suggest that exposures to apoptotic and necrotic ECs induce different patterns of chemokine synthesis and leukocyte adhesion in healthy ECs. These data indicate that CECs are not only markers of vascular damage but may induce proinflammatory signals in the endothelium.
[show abstract][hide abstract] ABSTRACT: Circulating endothelial cells (CECs) were first described over 30 years ago in smears of peripheral blood. Since then, more sophisticated techniques for CEC isolation have become available. In particular, immunomagnetic isolation and fluorescence-activated cell sorting (FACS) have been employed with success. We provide a short historical perspective and a comprehensive review on the subject. We review isolation and enumeration of CECs with an emphasis on CD146-driven immunomagnetic isolation and FACS. We describe, in great detail, advantages and pitfalls of both approaches and compare their specificity. Moreover, we provide a comprehensive list of clinical studies in this field and describe the possible clinical use of CECs. We also describe the phenotype of these cells and list typical surface markers. In addition, we review the phenotype of CECs and discuss mechanisms of detachment. We speculate about potential interactions between CECs and other cell subsets. We also describe other serum markers of endothelial damage and compare CECs with these markers. Finally, we highlight differences between circulating endothelial cells and endothelial progenitor cells. In summary, CECs must now be regarded as a sensitive and specific marker of endothelial damage. We emphasize that use of CECs in a clinical setting is on the horizon and pathogenetic clues may also be obtained.
[show abstract][hide abstract] ABSTRACT: Circulating endothelial cells (CECs) are a reliable marker of disease activity in a variety of vascular disorders. Damage to microvascular endothelial cells is a hallmark of thrombotic microangiopathy (TMA). The aim of this study is to identify and count CECs during the course of TMA and evaluate whether cell numbers may serve as a prognostic marker in patients undergoing plasma exchange.
Fifteen patients (8 women, 7 men) aged 31 to 66 years with TMA of different causes were studied before and after 4 sessions of plasma exchange. CECs were isolated by using anti-CD146-driven immunomagnetic isolation and counted after staining with Ulex Europaeus lectin-1.
Numbers of CECs were markedly elevated in all patients before treatment (64 to 672 CEC/mL; mean, 320 +/- 205 CEC/mL) compared with healthy controls (0 to 16 CEC/mL; mean, 6.4 +/- 4.2 CEC/mL; P < 0.001). Patients with a favorable outcome had significantly greater initial CEC levels (mean, 426 +/- 175 CEC/mL; P < 0.001), and cell numbers decreased significantly after 4 treatments of plasma exchange (mean, 101 +/- 53 CEC/mL; P = 0.001). Patients with disease unresponsive to plasma exchange presented with lower initial CEC levels (mean, 108 +/- 36 CEC/mL), and numbers failed to decrease after plasma exchange (mean, 114 +/- 57 CEC/mL; P = 0.827).
Markedly elevated numbers of CECs reflect severe and widespread endothelial damage in patients with TMA. Cell numbers at presentation and their degree of decrease after 4 sessions of plasma exchange could provide important prognostic clues.
American Journal of Kidney Diseases 11/2006; 48(4):564-70. · 5.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Circulating endothelial cells (CECs) have been identified as markers of vascular damage in a variety of disorders, such as myocardial infarction, vasculitis, and transplantation. CD146-driven immunomagnetic isolation has gained widespread use, but the technique is hampered by the lack of a definition of CECs and the absence of a consensus for their enumeration.
To evaluate several variables influencing immunomagnetic isolation of CECs, formulate a definition for CECs and propose a consensus protocol for their enumeration.
We devised a protocol based on CD146-driven immunomagnetic isolation and a subsequent confirmatory step with Ulex-Europaeus-Lectin-1 staining. In a multi-center effort, we evaluated the preanalytical and analytical phases of this protocol. We evaluated the effects of storage, anticoagulation and density centrifugation, and compiled previous experience with this technique.
Our protocol permitted unequivocal identification of CECs with acceptable reproducibility. There was an effect of storage time in that median cell numbers declined to only 87.5% of their baseline values during 24 h of storage at 4 degrees C. Recovery was lower with citrate than with ethylene-diamine tetra-acetic acid after 4 h of storage; density centrifugation was also associated with lower recovery. We provide a comprehensive list of technical recommendations and potential pitfalls. Finally, based on our experience with this protocol and a recent consensus workshop, we formulated a working definition for CECs.
Our work represents an important step toward consensus regarding the CECs. Our recommendations represent the experience of three major centers and should now be scrutinized by others in the field.
Journal of Thrombosis and Haemostasis 04/2006; 4(3):671-7. · 6.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: To evaluate numbers of circulating endothelial cells (CECs) in ANCA associated vasculitis and compare vasculitic relapse with limited granulomatous disease.
Sixteen patients with vasculitic relapse of ANCA associated vasculitis and 12 patients with limited granulomatous disease due to Wegener's granulomatosis (WG) were studied. Six patients with newly diagnosed vasculitic disease and six patients with vasculitis with infectious complications were also studied. Twenty two patients in remission were studied, as were 20 healthy controls. Counting of CECs was performed with anti-CD146 driven immunomagnetic isolation and staining with Ulex Europaeus lectin 1(UEA-1).
Patients with vasculitic relapse had markedly increased numbers of circulating endothelial cells (12-800 cells/ml, median 88 cells/ml) as did patients with newly diagnosed systemic vasculitis (20-216 cells/ml, median 56 cells/ml). Patients with limited granulomatous disease due to WG had only slightly increased cell numbers (4-44 cells/ml, median 20 cells/ml), which were similar to those of patients in remission (4-36 cells/ml, median 16 cells/ml). Numbers of CECs in patients with granulomatous disease were significantly lower than in those patients with relapse or new onset vasculitis (p<0.001). Cell numbers in patients with relapse and new onset vasculitis declined with immunosuppressive treatment. Patients with infection had 4-36 cells/ml (median 10 cells/ml). A cut off value of 20 cells/ml for a positive result yielded 64% specificity and 95% sensitivity for active systemic vasculitis; the positive predictive value was 63% and the negative predictive value 95%.
Markedly increased numbers of CECs discriminate active vasculitis from limited granulomatous disease and remission. These findings add further proof to the concept of CECs as a marker of ANCA associated small vessel vasculitis.
Annals of the Rheumatic Diseases 03/2006; 65(2):164-8. · 9.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: Churg-Strauss Syndrome (CSS) is characterized by allergic rhinitis, asthma and prominent blood and tissue eosinophilia. Although CSS can affect any organ system, isolated cardiac manifestation is a rare feature that is often characterized by rapidly progressive congestive heart failure. We present the case of a 48-year-old woman with acute dyspnoea and chest pain. Her past medical history was significant for asthma and frequently relapsing minimal-change glomerulonephritis. Echocardiogram and coronary angiography revealed cardiomyopathy and coronary small-vessel vasculitis in the presence of blood eosinophilia and elevated IgE. In the absence of infective agents, neoplastic diseases and further vasculitic manifestations, a flow cytometry-based analysis of markedly elevated endothelial microparticles supported the diagnosis of CSS. Cardiomyopathy resolved completely after initiation of immunosuppressive treatment with corticosteroids and cyclophosphamide pulses. Elevated endothelial, leukocytic and platelet-derived microparticles decreased during follow-up and closely paralleled vasculitic activity. Endothelial microparticles might be an additional tool to diagnose and monitor cases of suspected vasculitic cardiac involvement in CSS.