Detection and Validation of Circulating Endothelial Cells, a Blood-based Diagnostic Marker of Acute Myocardial Infarction

Background Circulating endothelial cells (CECs) are markers of vascular damage that have clinical relevance in many diseases, including acute myocardial infarction (AMI), and may be predictors of treatment responses. Herein, we investigated the diagnostic and prognostic value of CEC monitoring in AMI patients and a murine model. Methodology/Principal Findings CECs were defined as Hoechst 33342+/CD45−/CD31+/CD146+/CD133− in human blood samples and Hoechst 33342+/CD45−/CD31+/KDR+/CD117− in murine samples. To evaluate the validity and variability of our CEC detection system, peripheral blood samples of vascular endothelial growth factor-treated athymic nude mice and AMI patients were collected and subjected to intra-assay analysis. CEC detection by flow cytometry and real-time PCR were compared. Blood samples were obtained from 61 AMI patients, 45 healthy volunteers and 19 samples of the original AMI patients accepted one month treatment, via flow cytometry and expressed as a percentage of peripheral blood mononuclear cells. Results Our CEC detection method was validated and had limited variability. CEC concentrations were higher in AMI patients compared to healthy controls. One month post-treatment, CECs levels decreased significantly. Conclusions/Significance CEC levels may be useful as a diagnostic and prognostic biomarker in AMI patients.


Introduction
Circulating endothelial cells (CECs) are noninvasive markers of vascular damage, remodeling, and dysfunction [1], and considerable efforts have been directed at targeting the vascular components of malignant diseases. Currently, several methods are commonly used to identify CECs, including immunomagnetic isolation [2] and polychromatic flow cytometry [3]. CECs are present at very low levels in healthy subjects, whereas elevated levels have been reported in response to various pathological conditions, including acute myocardial infarction (AMI), coronary heart disease, infectious diseases, immunologic disorders, and cancers. [4,5,6] CECs can be used as a biomarker of these diseases, as they can potentially predict early responses to a course of treatment likely to benefit patients [7,8]. Further, multiple studies have reported that CEC concentration is a potential surrogate marker of anti-angiogenic drug activity [2,9,10].
Many CEC antigens have been monitored by flow cytometry using monoclonal antibodies, but there are no antibodies specific to antigens that can discriminate CECs from peripheral blood cells. For examples, CD146 is an endothelial-specific marker [11], but it is also expressed by some mesenchymal cells and a subpopulation of activated lymphocytes [12]. Hence, only a multiparametric, concurrent investigation using several antibodies can discriminate CECs from peripheral blood cells. The first step in such analyses is to exclude hematopoietic cells using the panhematopoietic marker CD45, and then confirm the endothelial nature of the remaining CD45-negative cells using two or more endothelial markers, such as CD146, CD31, or kinase insert domain receptor (KDR, also known as vascular endothelial growth factor receptor 2, VEGFR2).
Previous studies have established that CECs may be elevated in murine angiogenesis models, which can decrease following administration of antiangiogenic agents [13]. In murine models, the preclinical number of CEC correlates with angiogenic effects. Therefore, we hypothesize that CECs originating from endothelium sloughed into the circulation may increase after antiangio- genic administration. Because CECs occur during pathological conditions including vacuities, infection, and myocardial infarction [4,14,15,16], a method is needed to more accurately identify CEC populations to reflect the angiogenic microenvironment. CEC levels have also been investigated using a previously established model of VEGF-induced mice [17]. Hence, CECs present a useful marker for detecting angiogenesis or monitoring therapy responses that affect vasculature [18]. Myocardial infarction (MI) is a leading cause of death worldwide, and CEC levels are significantly elevated in AMI [4]; however, changes in CEC concentrations post-treatment have not been reported. Therefore, in the present study, we evaluated the use of CECs as a blood-based biomarker of cardiovascular diseases via flow cytometry and validated this method using known antigenic markers.

Materials and Methods
Endothelial Cell Line Culture

Animal Models
Ten athymic nude mice (8-10-week-old) were purchased from the Shanghai Laboratory Animal Center (Shanghai, China) and housed under pathogen-free conditions. All animal experiments were performed in accordance with institutional guidelines for animal care at Shanghai Jiao Tong University and approved by Animal Care and Use Committee of Shanghai Jiao Tong University. Every nude mouse was injected daily with 10 mg of recombinant human VEGF (National Cancer Institute, Biological Resources Branch, Frederick, MD, USA) for 5 days. Blood was collected by retro-orbital puncture and anticoagulated using ethylenediaminetetraacetic acid (EDTA).

Intra-assay Variability Analysis
To assess the technical variability of flow cytometry analysis, an intra-assay was performed on 5 VEGF-treated mice and 5 AMI patients. Briefly, peripheral blood samples were divided into 8 replicates and analyzed by flow cytometry. Intra-assay variability was calculated as the CV values of 8 replicates.

Measurement of CECs by Real-time PCR
Blood samples (2 ml) from 5 healthy mice and 5 VEGF-treated mice were obtained and 1 ml was subjected to flow cytometry analysis, and the other 1 ml from the same tube was mixed with 15 ml of erythrocyte lysis buffer (0.899% (w/v) ammonium chloride, 0.1% (w/v) potassium bicarbonate, and 0.0037% (w/v) EDTA, pH 7.3) and incubated for 10 min at room temperature (RT). After the samples were centrifuged for 5 min at 500 g, the lysis buffer was removed and the cell pellet was resuspended in 350 ml of RLT buffer (Qiagen, Hilden, Germany). RNA was isolated using the RNeasy Kit (Qiagen, USA) according to the manufacturer's instructions. Total RNA (2,500 ng) was reversetranscribed to complementary DNA (cDNA) using the Prime-Script 1st Strand cDNA Synthesis kit (TaKaRa Bio, Inc., Shinga, Japan) following the manufacturer's instructions. For the amplification of KDR (KDR sense 59-GAACCTGACTATCCG-CAGGG-39, and antisense 59-AGGAGCCAGAAGAA-CATGGC-39; GRAPDH sense 59-TGATGGG TGTGAACCACGAG-39, and antisense 59-ATCACGCCA-CAGCTTTCCAG-39), 1 ml of cDNA was added to SYBR Green PCR Master Mix (Qiagen, USA). PCR was performed on ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City. CA, USA) using the following thermal settings: one cycle of 10 min at 95uC, and 40 cycles of 15 s at 95uC, and 1 min at 60uC. Relative mRNA expression was calculated with the 2 2DDCt method [19], and the results were compared to the flow cytometry CEC analysis.

Daily Variability in CEC Percentage
To assess the daily variability in the relative CEC concentration of one AMI patient, peripheral blood was taken at different time points (0, 24, 48, and 72 h) and each sample was analyzed in 4 replicates. For the mouse study, all blood samples were collected at the same time and stored at RT due to bleeding affect the CEC concentrations. The samples were assayed at different time points (0, 24, 48, and 72 h) and each analyzed in 4 replicates.

Patients
The research was carried out according to the principles of the Declaration of Helsinki. Informed consents written by participants were obtained and this study was approved by Ethics Committee of the Shanghai Ninth People's Hospital, Shanghai JiaoTong  University School of Medicine. The patient data, which are contained within this article, were obtained by a hospital-based doctor at Shanghai Ninth People's Hospital and Ruijin Hospital, Shanghai JiaoTong University School of Medicine. Permission to use these data in this report has been obtained from all the subjects who participated in this study. This prospective study included consecutive AMI patients treated at the Ninth People's Hospital and Ruijin Hospital, Shanghai Jiaotong University School of Medicine (Shanghai, China). AMI was diagnosed via angiocardiography, biochemical markers, and cardiac enzyme tests.

CEC Analysis
The peripheral blood samples were collected from patients who met the AMI criteria. The percentage of CECs was determined by flow cytometry. One month post-treatment, blood samples were collected again from some of these original AMI patients.

Statistical Analysis
The results are presented as means 6 standard deviation (SD). The difference between two groups was analyzed using the twotailed Student's t-test and a confidence level of p,0.05 was considered statistically significant, p,0.01 was considered statistically very significant.

Measurement Consistency of CECs Detected by Flow Cytometry and Real-time PCR
The mean percentage of CECs in blood samples from normal and VEGF-treated mice was 0.256 and 3.104%, respectively ( Figure 3A). Relative KDR mRNA expression was measured in blood samples from VEGF-treated mice and healthy controls to characterize CEC concentrations. As shown in Figure 3B, VEGF treatment induced an 8.8-fold increase in KDR expression. The increased CEC percentage and relative KDR expression in VEGF-treated mice were in agreement.

Daily Variability in CEC Concentration
For the human study, peripheral blood samples were collected at different time points (0, 24, 48, and 72 h). As shown in Figure 4B, the percentage of CECs reached a maximum value at 24 h. For the mouse study, the blood samples were collected at the same time and stored at RT. The percentage of CECs was measured at different time points (0, 24, 48, and 72 h). As shown in Figure 4A, the percentage of CECs elevated quickly with time.

Elevated CEC Levels in AMI Patients
A total of 106 subjects were recruited for the present study between December 2011 and October 2012, which included 61 AMI patients and 45 age-matched healthy subjects. Table 1 summarizes the characteristics of the study groups. As shown in Figure 5, the percentage of CECs in AMI patients was significantly higher than that in healthy subjects ( Figure 5), which was consistent with the results of biochemical marker analysis and cardiac enzyme tests ( Table 2).

Changes in CEC Percentage Post-treatment
One month post-treatment, the peripheral blood of 19 AMI patients was collected again, and as the other 42 patients have left our hospital and we could not obtain further blood samples. As shown in Figure 6, the CEC percentage post-treatment decreased significantly compared with pre-treatment, whereas biochemical markers and cardiac enzymes were at normal levels.
CEC concentrations were significantly (8.8-fold) increased in VEGF-treated mice compared to normal controls. KDR (VEGFR2) is a marker of CEC [23], detected by real-time PCR was consistent with CEC percentage detected by flow cytometry, suggesting that our detection system was valid.
Cardiovascular disease is a leading cause of morbidity and mortality worldwide and methods for assessing endothelial function are well established. The measurement of immunologically defined CECs in peripheral blood is gaining ground as an important technique for assessment of endothelial injury [1,4]. In this study, we found that CEC levels were elevated in peripheral blood samples from AMI patients; hence, CECs can act as a blood-based biomarker of cardiovascular diseases and pose a good target for MI pharmaceutical development.
CECs may also have clinical relevance in many other diseases, as CEC concentration was increased in cancer patients and correlated with tumor progression [24,25,26]. Further, CEC quantification can identify patients who might benefit from angiogenesis inhibitors and be used to monitor treatment response [27,28]. In the current study, CEC concentrations decreased significantly 1 month post-treatment compared with pre-treatment, but still remained higher than in healthy controls. However, biochemical markers and cardiac enzymes were at normal levels, indicating that CECs present a useful prognostic marker.
In summary, the CEC concentration in peripheral whole blood is quite low; therefore, the interpretation of results should be made cautiously. Since the performance of the standardized flow cytometry method for CEC quantification was stable, it is suitable for use in clinical applications. In murine models, CEC levels increased following VEGF-treatment compared to normal control mice. Therefore, CEC levels provide information specific to angiogenesis and present a blood-based biomarker detection system to clinically monitor drug efficacy. However, the role of endothelial markers in cardiovascular disease remains to be addressed in future mechanistic and prospective clinical studies using other tissues. Also, new markers and methods must be developed to more accurately measure the angiogenic process. Furthermore, anti-angiogenesis therapy can limit angiogenesis, which can be monitored via CEC concentrations using the immunomagnetic separation method [2]. In future studies, CEC monitoring can be used to monitor targets of anti-angiogenesis therapies.