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ORIGINAL ARTICLE |
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Year : 2018 | Volume
: 31
| Issue : 4 | Page : 1435-1441 |
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Assessment of circulating endothelial cells in preeclamptic and normotensive pregnancies
Waleed M Fathy1, Ahmed A Sonbol1, Wael G Al-Damaty2, Shymaa A El Askary3, Bahaa El-Deen W. El-Aswad1
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Al Minufiyah, Egypt 2 Department of Obstetrics and Gynecology, Faculty of Medicine, Menoufia University, Al Minufiyah, Egypt 3 Department of Medical Microbiology and Immunology, Faculty of Medicine, Menoufia University, Al Minufiyah, Egypt
Date of Submission | 28-Jun-2017 |
Date of Acceptance | 07-Aug-2017 |
Date of Web Publication | 14-Feb-2019 |
Correspondence Address: Bahaa El-Deen W. El-Aswad Medical Microbiology and Immunology Department, Faculty of Medicine, Menoufia University, Shebin Elkom, 32511 Egypt
Source of Support: None, Conflict of Interest: None | Check |
DOI: 10.4103/mmj.mmj_467_17
Objective The aim of this study was to assess the level of circulating endothelial cells (CECs) in women with preeclampsia (PE) in comparison with normotensive healthy pregnant controls. Background PE is a medical disorder that may affect pregnant women after 20th week of gestation, and in this condition, the patients experience new-onset hypertension and new-onset proteinuria or organ dysfunctional. There is placental ischemia with systemic maternal endothelium dysfunction. CECs are mature cells that shed from the endothelium in various diseases associated with vessel damage, and they are characterized as being CD146+ and CD45− cells. Patients and methods The current study included 30 patients with preeclampsia and 30 normotensive pregnant women as the control group. All the participants attended Department of Obstetrics and Gynecology, Faculty of Medicine, Menoufia University Hospitals. They were subjected to collection of demographic data (age and length of gestation), general clinical examination, in addition to undergoing some routine laboratory investigations. Flow cytometry was applied for quantification of CECs using both mouse antihuman fluorescein isothiocyanate-conjugated CD146 antibody and mouse antihuman phycoerythrin conjugated CD45 antibody. Results The CEC counts were significantly (P < 0.001) lower in women with PE (4026.67 ± 1464.5/5 × 106 peripheral mononuclear cells) in comparison with the healthy pregnant ones (9281.33 ± 1764.06/5 × 106 peripheral mononuclear cells). Additionally, CD146 expression strength (mean) was significantly (P < 0.001) higher in healthy pregnant women (93.81 ± 27.57) compared with that of those with PE (43.51 ± 14.47). Conclusion The number of CECs decreased significantly in patients with PE in comparison with normotensive healthy pregnant women.
Keywords: endothelial cells, flow cytometry, preeclampsia, pregnancy
How to cite this article: Fathy WM, Sonbol AA, Al-Damaty WG, El Askary SA, W. El-Aswad BE. Assessment of circulating endothelial cells in preeclamptic and normotensive pregnancies. Menoufia Med J 2018;31:1435-41 |
How to cite this URL: Fathy WM, Sonbol AA, Al-Damaty WG, El Askary SA, W. El-Aswad BE. Assessment of circulating endothelial cells in preeclamptic and normotensive pregnancies. Menoufia Med J [serial online] 2018 [cited 2024 Mar 29];31:1435-41. Available from: http://www.mmj.eg.net/text.asp?2018/31/4/1435/252062 |
Introduction | | |
Preeclampsia (PE) is a medical disorder affecting pregnant women beyond the 20th week of gestation. It presents with new-onset hypertension, in addition to either new-onset proteinuria or terminal organ dysfunction[1],[2]. PE could be classified according to onset of clinical symptoms into early, before the 34th week of gestation; late, after the 34th week of gestation; or according to its severity, mild or late PE[3],[4]. It affects 5–8% of all pregnancies worldwide, and it may lead to severe morbidity or even mortality of mother and their fetuses[5].
Several theories have been suggested to explain pathogenesis of PE; however, this issue is still not fully clarified[4]. The most accepted theory proposes that the cytotrophoblast does not invade the spiral miometrial arteries correctly leading to impairment of the vessels transformation, so these vessels remain small causing placental ischemia resulting in release of vasoactive factors that may lead to systemic maternal endothelium dysfunction[6],[7],[8].
Circulating endothelial cells (CECs) are mature cells that purely originate from the inner lining of vessels in response to endothelial injury because of mechanical agents, infection, activation of apoptosis, enhanced proangiogenic factors at the expense of antiangiogenic ones, inflammatory cytokines, or under the effect of proteases[9],[10],[11],[12].
The count of CECs increases in wide range of disorders accompanied with vascular damage, such as myocardial infarction[13], cerebrovascular injury[14], diabetes mellitus[15],[16], acute respiratory distress syndrome[17], systemic lupus erythematosus[18], sepsis[19], and acute myeloid leukemia[20]. So, the number of CECs may strongly reflect the condition of the endothelial lining, and it is suggested to be an attractive marker reflecting the extent of the vessel damage[21],[22].
These cells express specific endothelial cell phenotype markers, like CD146, Willebrand factor, or CD31; however, they are negative for CD45, a hematopoietic cells marker, or CD133, a progenitor cell marker[23],[24]. These cells are quantified most widely by immunomagnetic techniques or using multicolor flow cytometry (FCS)[25].
Few reports have studied the potential role of CECs in the pathogenesis of PE[26],[27]. Herein, the current study is aimed at assessing the level of CECs in women with PE compared with their normotensive counterparts matched for gestational age in Menoufia Governorate, Egypt.
Patients and Methods | | |
This study included 30 patients diagnosed as having PE, in addition to 30 maternal age-matched and gestational age-matched healthy normotensive pregnant females as the control group. All participants attended the Department of Obstetrics and Gynecology, Faculty of Medicine, Menoufia University Hospitals during the period from January 2016 to January 2017. The ethics committee of Faculty of Medicine, Menoufia University, approved this study, and written consent was obtained from each participant.
All participants were subjected to recording their demographic data (age and length of gestation), general clinical examination, measuring blood pressure, and undergoing some laboratory investigations, which included 24 h proteinuria, complete blood count (CBC), serum creatinine level, serum urea level, fasting blood sugar (FBS), and prothrombin time (PT).
PE cases were diagnosed when they had newly onset high blood pressure (systolic ≥140 mmHg or diastolic ≥90 mmHg) and newly onset proteinuria (≥300 mg/24 h) after the 20th week of pregnancy. Cases having cardiovascular, renal, infection, inflammatory, diabetes mellitus, autoimmune, or neoplastic conditions were excluded from this study.
From each participant, 15 ml of blood was obtained under aseptic technique. Overall, 10 ml of blood was poured into EDTA containing tube for measuring CECs, 1 ml was put into another EDTA having tube to perform CBC, 1.8 ml venous blood was transferred into a clean tube containing 0.2 ml Na citrate used for PT measuring, and the remaining of the blood was poured into plain glass tube for serum urea and creatinine level measuring. Urine samples were collected in sterile container over a period of 24 h, and the protein content, expressed as mg/h, was measured.
CBC was analyzed by Advia 2120 automated blood counter (Siemens Healthcare Diagnostics, Tarrytown, New York, USA). Serum creatinine level, serum urea level, and FBS were measured by autoanalyzer Synchron CX5 (Beckman Coulter Inc., Brea, California, USA). PT was measured using rabbit brain thromboplastin reagent (Spinreact, Girona, Spain) with manual method according to the manufacturer's instructions. Quantitation of protein in collected urine/24 h was done using colorimetric method, where protein reacts with pyrogallol red and molbdata in acid solution to form a red complex (BioSystems, Barcelona, Spain).
Counting of circulating endothelial cells by using flow cytometry
Overall, 10 ml of blood was mixed with equal volume of PBS (pH 7.4), layered on Ficoll reagent (1077 g/ml; Sigma-Aldrich, Missouri, St. Louis, MO, USA), and centrifuged at 400 g for 20 min (min). The buffy coat was recovered, mixed with 10 ml of PBS, and centrifuged at 300 g for 10 min; thereafter, the cell pellet was suspended in 1 ml PBS adjusting the count of peripheral mononuclear cells (PBMCs) to be 1 × 106/ml.
Overall, 200 μm of the isolated PBMCs was incubated with 20 μl of each of mouse antihuman fluorescein isothiocyanate-conjugated CD146 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) and mouse antihuman phycoerythrin conjugated CD45 antibody (Miltenyi Biotec) for 30 min in the dark. These monoclonal antibodies identified CD146+ and CD45− cells. An isotype control antibody was used to discriminate between negative and positive fluorescence stain and to distinguish between nonspecific background signal from specific antibody one. Stained samples were analyzed with FACSCalibur flow cytometer (BD Biosciences, San Jose, California, USA). Internal complexity (SSC) versus a size (FSC) dot plot was developed, and then a region of population of the cells having mononuclear cells was drawn for data analysis.
For each sample, 100 000 PBMCs were analyzed by FCS to assess the number of CECs and the result was expressed as number of CECs per 5 × 106 PBMCs.
Statistical analysis
Statistical package for the social sciences (version 20; SPSS Inc., Chicago, Illinois, USA) was used for collection, tabulation, and analysis of the results statistically. The descriptive statistics were expressed in number, percentage, mean, and SD. Student's t-test was applied for comparison of quantitative variables between two groups of normally distributed data. Mann–Whitney test was used for comparison of quantitative variables between two groups of not normally distributed data, and Pearson's correlation was used to show correlation between two continuous normally distributed variables. P value less than 0.05 was considered significant.
Results | | |
FCS analysis of CECs is represented in [Figure 1]. There were no significant differences between both groups for maternal age, gestational age, or FBS (P = 0.65, 0.63 or 0.72, respectively). Moreover, laboratory parameters involving platelet count (P = 0.07), creatinine level (P = 0.06), urea level (P = 0.065), PT (P = 0.63), red blood cells count (P = 0.058), hemoglobin (P = 0.28), and hematocrit percentage (P = 0.15) did not differ significantly in patients with PE in comparison with healthy pregnant controls [Table 1]. | Figure 1: Representative flow cytometry analysis of circulating endothelial cells (CECs). Healthy pregnant controls: (a) unstained autocontrol sample, (b) gating on peripheral mononuclear cells (R1), and (c) mature CECs (negative CD45 and positive CD146). Patients with preeclampsia: (d) unstained autocontrol sample, (e) gating on peripheral mononuclear cells (R1), and (f) mature CECs (negative CD45 and positive CD146).
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The systolic and diastolic blood pressures in patients with PE (163 ± 13.23 and 98.33 ± 7.11 mmHg, respectively) were higher than that in controls (116.13 ± 9.14 and 74.5 ± 6.86 mmHg, respectively) with significant values (P < 0.001 and <0.001, respectively). The collected protein in 24 h in the urine in women with PE group (579.73 ± 134.32 mg/24 h) was statistically significant (P < 0.001) more than that of the pregnant control ones (125.36 ± 19.20 mg/24 h) [Table 1].
The CECs numbers decreased significantly (P < 0.001) in women with PE (4026.67 ± 1464.5/5 × 106 PBMCs) in comparison with the healthy pregnant women (9281.33 ± 1764.06/5 × 106 PBMC) [Table 2] and [Figure 1]. Moreover, strength of CD146 expression (mean) was higher significantly (P < 0.001) in controls (93.81 ± 27.57) than in patients with PE (43.51 ± 14.47) [Table 2] and [Figure 2]. | Table 2: Circulating endothelial cells counts and strength of CD146 expression (mean) of the studied groups
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| Figure 2: Circulating endothelial cells count of patients with preeclampsia and healthy pregnant control groups.
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CEC count showed nonsignificant negative correlation with maternal age (r=−0.17; P = 0.35), systolic blood pressure (r=−0.13; P = 0.48), diastolic blood pressure (r=−0.06; P = 0.71), urea level (r=−0.06; P = 0.74), FBS (r=−0.19; P = 0.29), and PT (r=−0.07; P = 0.69). Also, the cells nonsignificantly positively correlated with gestational age (r = 0.18; P = 0.31), proteinuria, (r = 0.02; P = 0.91), creatinine level (r = 0.04; P = 0.79), red blood cells (r = 0.06; P = 0.74), hemoglobin (r = 0.07; P = 0.69), hematocrit percentage (r = 0.21; P = 0.24), and platelet (r = 0.01; P = 0.95) [Table 3] and [Figure 3]. | Table 3: Correlation between circulating endothelial cells count and demographic and laboratory data of patients with PE
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| Figure 3: Strength of CD146 expression (mean) of patients with preeclampsia and healthy pregnant controls groups.
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Discussion | | |
It is known that formation of new blood vessels and enhanced endothelial function accompany normal pregnancy; however, PE causes prominent systemic impairment of maternal endothelial function which may be noticed for several years[4],[28]. In this work, the count of CECs was evaluated using FCS among women with PE. Evidences from many reports denote the efficacy of CECs as a reliable indicator for the endothelial malfunction or injury in many diseases[13],[22],[29],[30].
Generally speaking, there are few reports that have evaluated the level of CECs in PE. Herein, we applied FCS for enumeration of CECs because FC has been proven to possess a high sensitivity for determination of CECs phenotypic markers[27],[31].
To our updated knowledge, the current report is the first one that evaluated CECs number in Egyptian patients. Unexpectedly, our patients with PE showed significantly lower CECs count than that of healthy pregnant controls. Our result was in accordance with Heimrath et al.[32] who reported that the group of patients with pregnancy-induced hypertension, which included patients with PE, had 1.28 × 103/1 ml CECs, which was nearly twice as lower compared with normotensive healthy controls (2.77 × 103/1 ml). Heimrath et al.[32] used FCS for quantification of the cells in this study.
Also, Strijbos et al.[33] found that CECs count, detected by anti-CD146 antibody conjugated with ferrofluids, in patients with PE (median count = 5.3 cells/ml) did not differ significantly (P = 0.21) from that of the normotensive controls (median count = 3.5 cells/ml); however, serum E-selectin and endoglin, markers for endothelial dysfunction, and thrombomodulin, an endothelial injury marker, all were higher in the PE group than normal pregnancy ones.
We did not find any significant correlation between CEC numbers and any demographic, clinical, or laboratory parameters, and this was in accordance with Strijbos et al.[33].
However, our result was in contrast with other many studies that reported higher significant CECs count in PE in comparison with normotensive pregnant controls. Canbakan et al.[34] reported significantly higher CECs count in women with PE (13.2 ± 5.2 cells/ml) in comparison with healthy pregnant ones (5.2 ± 1.4 cells/ml), healthy women (4.0 ± 1.8 cells/ml), and even nonpregnant women with hypertension (6.9 ± 0.8 cells/ml), using anti-CD146 antibody, and the cells numbers correlated positively with serum homocysteine levels.
Grundmann et al.[26] recorded markedly significant elevated CECs numbers in women with PE (median 88 cells/ml), when compared with either normal pregnancies (median 16 cells/ml) or healthy nonpregnant women (12 cells/ml), and after delivery, CECs returned to their normal count rapidly. They exploited anti-CD146 immunomagnetic isolation with Ulex Lectin staining.
Karthikeyan et al.[31] recoded an increment of the CECs numbers in a group of pregnant women with hypertension in comparison with either healthy pregnant women (lower by twice-fold) or healthy nonpregnant women (lower by four folds). These researchers measured CECs by immunomagnetic isolation with anti-CD146 monoclonal antibody coated beads.
Mehrabian et al.[35] used immunomagnetic bead method for quantification of CECs and found that there was significant increment of the cells in women with PE (the cutoff number was 6.5 cells/ml) more than normotensive pregnant women, and level of serum E-selectin was higher in PE group.
Additionally, Anim-Nyame et al.[36] found that CECs number increased significantly in women with PE (24 ± 2.6 cells/ml) when compared with normotensive pregnancies (12.7 ± 1.9 cells/ml); moreover, insulin resistance was more in women with PE than normal pregnancy. They used immunomagnetic bead separation for CECs quantitation.
The CECs number was higher significantly in women having an active PE (9.9 ± 7.9 cells/ml) than women with a history of PE (3.4 ± 4.0 cells/ml) or healthy pregnant women (3.0 ± 4.1 cells/ml); moreover, the patients with active PE had significantly higher soluble vascular cell adhesion molecule-1 and soluble E-selectin[37].
Recently, Szpera-Goździewicz et al.[27] reported that CECs count in the blood samples of patients with PE (94 ± 54 cells) was nearly similar to that of women with chronic hypertension (95 ± 29 cells), and both were significantly higher than that in healthy pregnant women (56 ± 24 cells).
In our work, this unexpected low CEC count in PE could be related to several factors alone or in combination. Most of patients with PE enlisted in our study might have mild form of PE, having minimal endothelial damage, but we did not categorize these patients according to severity of the disease. Strijbos et al.[33] suggested that the damage of endothelial lining that is characterized by elevated numbers of CECs, in PE, was less prominent than endothelial dysfunction that is associated with minimal endothelial injury. In addition, the duration of PE may cause discrepancy in results as suggested by Strijbos et al.[33]; however, they did not find a significant correlation between CECs number and gestational age of their cases. Also, Strijbos et al.[33] observed a large SD of CEC in their groups leading to statistically nonsignificant difference.
Moreover, there is no consensus regarding the standard steps of method used for CECs counting during PE until now, and this issue may explain partly the difference in the results[27]. The most commonly used methods are immunomagnetic bead separation[26],[28] and FCS[27],[32]. Grundmann et al.[26] isolated CECs with anti-CD146 antibody, and then stained the cells with Ulex Lectin; this may falsely identified other cell types overestimating CECs.
Moreover, CECs counts are interpreted in two different ways of expression either CECs number per 1 ml of blood[27],[33], or the number in a known number of mononuclear cells[32], and this again may lead to difficulty in comparison among different studies. In healthy individuals, CEC levels are around 20 cell/ml[38],[39], whereas in PE, CEC levels are 5.3–88 cells/ml[26], but it may reach to 204 cells/ml in vasculitis cases[21].
Lastly, concurrent undiagnosed other pathological conditions, like insulin resistance, may decrease CECs counts[40]. We suggest that a considerable number of CECs that shed from the injured vessels during PE in our study were disintegrated and may show loss of CD146, so they might have been underestimated.
Conclusion | | |
CECs number count decreased in patients with PE in comparison with healthy pregnant women. It is recommended to conduct similar study with a larger number of participants and to assess the number of CECs in longitudinal pattern before PE development through pregnancy period.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]
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