Uta Erdbruegger

Isolation and enumeration of circulating endothelial cells by immunomagnetic isolation: proposal of a definition and a consensus protocol

Summary.  Background: 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. Aim: To evaluate several variables influencing immunomagnetic isolation of CECs, formulate a definition for CECs and propose a consensus protocol for their enumeration. Methods: 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. Results: 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 °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. Conclusion: 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.

Detection of circulating microparticles by flow cytometry: influence of centrifugation, filtration of buffer, and freezing

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.

Rituximab as rescue therapy in anti-neutrophil cytoplasmic antibody-associated vasculitis: a single-centre experience with 15 patients

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Acute Tubular Injury in Protocol Biopsies of Renal Grafts: Prevalence, Associated Factors and Effect on Long-Term Function

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.

Circulating Endothelial Cells: Markers and Mediators of Vascular Damage

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.

Impact of CMV infection on acute rejection and long-term renal allograft function: a systematic analysis in patients with protocol biopsies and indicated biopsies

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Endothelial-derived thrombospondin-1 promotes macrophage recruitment and apoptotic cell clearance

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.

Circulating endothelial cells and stroke: influence of stroke subtypes and changes during the course of disease.

BACKGROUND 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. METHODS 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). RESULTS 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. CONCLUSIONS CECs are markers of endothelial damage and/or repair in stroke. Differences during the course of disease are likely to reflect different pathophysiology.

Imaging Flow Cytometry for the Characterization of Extracellular Vesicles.

Extracellular Vesicles (EVs) are potent bio-activators and inter-cellular communicators that play an important role in both health and disease. It is for this reason there is a strong interest in understanding their composition and origin, with the hope of using them as important biomarkers or therapeutics. Due to their very small size, heterogeneity, and large numbers there has been a need for better tools to measure them in an accurate and high throughput manner. While traditional flow cytometry has been widely used for this purpose, there are inherent problems with this approach, as these instruments have traditionally been developed to measure whole cells, which are orders of magnitude larger and express many more molecules of identifying epitopes. Imaging flow cytometry, as performed with the ImagestreamX MKII, with its combination of increased fluorescence sensitivity, low background, image confirmation ability and powerful data analysis tools, provides a great tool to accurately evaluate EVs. We present here a comprehensive approach in applying this technology to the study of EVs.

Circulating endothelial cells and endothelial progenitor cells after angioplasty: news from the endothelial rescue squad

In the 19th century, Friedrich von Recklinghausen discovered that blood vessels possess a lining [1]. It has been estimated that 1.2 trillion of these cells cover a surface of 400 m. They are not, as Recklinghausen had believed, a mere tapestry . Instead, endothelial cells regulate vascular tone via nitric oxide, ensure the homoeostasis of coagulation, and participate in immune events via complex interactions with circulating cell subsets [2]. Endothelial dysfunction and damage have been the focus of investigators during the past decade and various approaches have been employed. Flow-mediated dilatation of the brachial artery has gained widespread use but the technique remains cumbersome. Endothelial damage can be assessed by soluble markers, such as thrombomodulin or von Willebrand factor, circulating endothelial cells (CECs) [3] and, more recently, endothelial microparticles [4]. Likewise, repair of endothelial damage has been studied ever since Jeffrey Isner and colleagues at Tufts University described bone marrow-derived endothelial progenitor cells (EPCs) [5]. Unfortunately, these cells still lack a clear-cut phenotype as reviewed elsewhere [6]. In this issue of the Journal, Bonello and colleagues from Marseille [7] report a remarkable study on CECs and EPCs in patients with stable coronary artery disease who underwent percutaneous catheter intervention (PCI). It was already known that numbers of CECs are elevated after myocardial infarction (MI) [8]. In fact such studies had already been done in the 1970s, albeit with primitive methodology [9]. Furthermore, EPCs have been demonstrated previously after MI [10]. What additional information does this study provide? Bonello and co-workers studied a cohort of patients with stable coronary artery disease and not with acute coronary syndromes. It is remarkable that elevated numbers of CECs are also found after angioplasty in stable coronary artery disease. These cells must derive from endothelial damage by the catheter procedure itself, as cell numbers had been normal at baseline. Immunomagnetic isolation is a very sensitive approach as the technique was originally devised to isolate circulating tumor cells. Flow cytometry may also have its advantages although a direct comparison between the two techniques is not available so far. Mechanisms of endothelial cell detachment remain simple whenmechanical force is themost likely cause, as in the present study. In contrast, we do not know the sequence of events in inflammatory disorders [11]. Factors that protect against endothelial detachment, such as cadherins, integrins and fibronectin, exist as well [11]. Little, if anything, is known about the kinetics of CEC clearance from peripheral blood. Bonello demonstrated a peak of CEC values after 6 h while cell numbers declined to baseline after 1 week. Such data need further corroboration, probably from animal models. Further studies should elucidate the fate of CECs, mechanisms of clearance and interactions with other cells [11]. Figure 1 summarizes the concept of endothelial cell detachment. Bonello and co-workers next analyzed hematopoietic progenitor cells and EPCs, and found an increase in progenitor cells as early as at the end of the procedure. It is amazing to observe this very timely reaction of EPCs as an endothelial rescue squad . Various mechanisms have been proposed to explain how EPCs are alerted to ongoing endothelial damage. At present, vascular endothelial growth factor (VEGF) is best characterized. Elevated serum levels of VEGF have been demonstrated as a sequel to ischemic injury and VEGF has been shown to mobilize EPCs from bone marrow [5]. More recently, the discovery that erythropoietin (EPO) regulates EPCs [12] has sparked considerable interest, not only among nephrologists who use this drug on a daily basis. These observations provided a tantalizing opportunity to manipulate the EPC system in vascular disease. Studies into the use of EPO in stroke and MI are currently under way. Administration of EPO would be much less cumbersome than the direct application of stem cells as described after MI [13]. More regulators of EPCs are currently emerging and others still await Correspondence: Alexander Woywodt, Division of Nephrology, Department of Medicine, Hannover Medical School, Carl-NeubergStrasse 1, 30625 Hannover, Germany. Tel.: +49 511 532 6319; fax: +49 511 552366; e-mail: woywodt. alexander@mh-hannover.de Journal of Thrombosis and Haemostasis, 4: 976–978