Context.—Systemic lupus erythematosus (SLE) is associated with an increased risk of atherosclerosis; endothelial dysfunction represents the first step in its pathogenesis.
Objective.—To assess endothelial dysfunction in SLE by circulating endothelial cells (CECs) and to characterize SLE-specific factors that contribute to its appearance.
Design.—Case-control study was conducted on 60 subjects, divided into 2 groups: group A (30 patients with SLE) and group B (30 healthy sex- and age-matched controls). Total cholesterol, triglycerides, antinuclear antibodies, anti–double-stranded DNA antibodies, and C3 were determined in all patients. Systemic lupus erythematosus activity was assessed using the SLE Disease Activity Index. Endothelial function was assessed by means of flow-mediated dilation of the brachial artery using B-mode ultrasonography and relative quantification of CD 146 mRNA by real-time polymerase chain reaction.
Results.—The group of SLE patients was formed of 20 females and 10 males, with a mean age of 31.16 ± 9.69 years. The values of SLE-specific tests and SLE Disease Activity Index were represented by anti–double-stranded DNA antibodies 160 ± 40.5, C3 68.91 ± 11.91 mg/dL, total cholesterol 188.66 ± 49.63 mg/dL, triglycerides 143.41 ± 46.26 mg/dL, and SLE Disease Activity Index 12.66 ± 3.70. Values for flow-mediated dilation were 8.85% ± 2.02% (group A) and 20.33% ± 6.19% (group B), P < .001, and CECs were 300 ± 40.5 μL−1 blood (group A) and 10 ± 2.5 μL−1 blood (group B). The statistical analysis showed a strong inverse correlation between CECs and SLE Disease Activity Index, a strong correlation between CECs and C3, a strong correlation between CECs and anti–double-stranded DNA antibodies, and a moderate inverse correlation between CECs and total cholesterol.
Conclusion.—Endothelial dysfunction is present in SLE patients even in the absence of traditional cardiovascular risk factors due to disease activity.
Systemic lupus erythematosus (SLE) is an autoimmune disease with a wide range of clinical manifestations.1 Despite the improvement of therapeutic regimes, the morbidity and mortality pattern in patients with this disease remains: in the first part of disease evolution, mortality is due to severe infections or to disease activity; later, it is caused by accelerated atherosclerosis and its consequences.2
In healthy subjects, the endothelium is not a simple physical barrier between the blood flow and the underlying tissues. This structure has many functions, such as continuous regulation of vascular tone, leukocyte adhesion, and maintenance of the balance between thrombotic and anticoagulant properties of the blood.3 When these functions of the endothelium are affected, endothelial dysfunction appears. Endothelial dysfunction is considered the first step in the atherogenic process; it has been identified even in patients with SLE without cardiovascular risk factors.4 Endothelial dysfunction in SLE is produced by the clustering of traditional risk factors, adverse effects of treatment, and SLE itself as an independent risk factor.5,6 Systemic inflammation, autoantibodies directed to double-stranded DNA, ribonucleoproteins, endothelial cells, phospholipids, activated complement products, lupus nephropathy, and dyslipidemia represent some factors related to SLE that contribute to the appearance of endothelial dysfunction.7,8
In the pathogenesis of SLE, genetic as well as environmental and hormonal factors are considered to be responsible for the development of multiple immunologic phenomena. Recently, the processes of angiogenesis and vasculogenesis and their dysfunction have been considered in the pathogenesis of SLE. Vascular lesions seem to be responsible for the cutaneous, nephritic, cardiovascular, and gastrointestinal symptoms.9 Atherosclerotic vascular events are major contributors to the clinical presentation of late-stage lupus.10
Endothelial cells may appear in the circulation by detaching from activated or damaged vessels. An increase of circulating endothelial cells (CECs) is described in several pathologic conditions that involve vascular injury or instability, such as myocardial infarction, infectious vasculitis, and cancer.11 These CECs are mostly viable and still exhibit proliferative capacity despite their terminal differentiation.12
The study of endothelial injury is difficult because of inaccessibility of the endothelium in humans. However, CECs may serve as a new marker for microvascular injury.13 Circulating endothelial cells are thought to be mature cells that have detached from the intimal monolayer in response to endothelial injury. Several possibilities can be considered for the mechanism responsible for endothelial detachment. It might be due to apoptosis, mechanical dislodgment of cells, or proteolysis of subendothelial matrix proteins, or it might be a consequence of complement-dependent injury.14,15 An increase in CEC number has been recently detected in different diseases, most of which are characterized by prominent vascular pathology, such as acute coronary syndromes,16 sickle cell anemia, antineutrophilic cytoplasmic antibody–associated small vessel vasculitis, systemic sclerosis, and SLE.17
Numbers of CECs reflect the extent of the endothelial lesion. High cell counts have been observed in diseases with widespread vascular damage, such as sickle cell disease or vasculitis. In contrast, localized damage may be seen in patients undergoing coronary angioplasty, and low cell numbers have been observed in this setting. In healthy subjects, renewal of the endothelial layer takes place at a low replication rate of 0%–1% per day. Therefore, detection of CECs in a healthy adult is a rare event; about 0–12/mL blood is considered normal.18
The quantification of CECs as an index of endothelial damage/injury may have advantages over plasma levels of endothelial perturbation, such as von Willebrand factor, soluble E-selectin, or thrombomodulin. Although these plasma endothelial markers are relatively easy to measure, there is uncertainty about whether a particular marker represents endothelial activation, endothelial dysfunction, or endothelial damage.19
CD146 (S-Endo-1) was initially identified as a marker of tumor progression and metastasis formation in human melanoma.20,21 CD146 is homologous to several cell adhesion molecules and belongs to the immunoglobulin superfamily containing 5 extracellular immunoglobulin-like domains, a single transmembrane domain, and a short cytoplasmic domain.2 Beside its expression in malignant melanocytes, CD146 is constitutively expressed in all endothelial cells, irrespective of anatomic localization.22 There has been lack of consensus on the appropriate method for CEC measurement. This finding has raised considerable questioning of the reliability of the flow cytometric method for CEC measurement and searching for a new method for detection of CECs.8
Although CECs have been documented in autoimmune conditions, including lupus and vasculitis, the relationship of endothelial cell injury/apoptosis with development of vascular dysfunction in SLE has not been prospectively investigated. We hypothesized that enhanced CECs in SLE correlate with increased disease activity. The aim of this study is the assessment of endothelial dysfunction in SLE patients by quantification of CECs using real-time polymerase chain reaction (PCR) and the characterization of SLE-specific factors that contribute to its appearance.
MATERIALS AND METHODS
Study Design
This study was carried out as a case-control study. The study was performed on 2 groups of subjects: group A, formed by 30 patients with SLE, and group B, formed by 30 healthy sex- and age-matched controls. The diagnosis of SLE was established based on American College of Rheumatology criteria. The procedures followed were in accordance with the ethical standards of our local ethics committee.
The SLE patients were from the medical outpatient clinic and or were inpatients of the physical medicine, rheumatology, and rehabilitation department, nephrology unit, and internal medicine department of Suez Canal University Hospital, Ismailia, Egypt. The patients were selected for the study according to certain inclusion and exclusion criteria. Control subjects included 30 volunteers matching age, sex, and vascular risk factors of the study group. They were recruited from healthy blood donors. Informed consent was obtained from all participating patients and controls. Participants' SLE activity was assessed using the SLE Disease Activity Index. Endothelial function was assessed by means of flow-mediated dilation (FMD) of the brachial artery using B-mode ultrasonography, and CD146 mRNA was measured by real-time PCR.
Methods
A 5-mL venous blood sample was withdrawn and divided as follows: 1 mL blood on ethylenediaminetetraacetic acid for complete blood cell count, 1 mL blood on ethylenediaminetetraacetic acid for assessment of CECs through total RNA extraction for mRNA CD146 expression by real-time PCR, and 3 mL left to be clotted at room temperature for 30 minutes, then centrifuged and separated; serum was divided to perform both routine and special laboratory investigations. A complete blood cell count using a Sysmex K-80 cell counter (Sysmex, Kobe, Japan); assessment of C-reactive protein (enzyme-linked immunosorbent assay) and fasting blood sugar; liver function tests that included serum aspartate transaminase, serum alanine transaminase, and albumin; kidney function tests that included blood urea and serum creatinine and lipid profile; and assessments of total cholesterol (normal up to 200 mg/dL; to convert to millimoles per liter, multiply by 0.0259), and triglycerides (normal range 50–150 mg/dL; to convert to millimoles per liter, multiply by 0.0113), complements (C3, C4) by radial immunodiffusion, and anti–double-stranded DNA antibodies by enzyme-linked immunosorbent assay and rheumatoid factor were performed.
Detection of CECs (CD146 mRNA) by Real-Time PCR
A total of 0.5 mL ethylenediaminetetraacetic acid blood was mixed with 1.4 mL of erythrocyte lysis buffer (0.899% [wt/vol] ammonium chloride, 0.1% [wt/vol] potassium bicarbonate, and 0.0037% [wt/vol] ethylenediaminetetraacetic acid, pH 7.3) and incubated for 10 minutes at room temperature. After 10 minutes centrifugation at 500g the buffer was removed and the cell pellet was resuspended in 350 µL RLT buffer (Qiagen, Mississauga, Canada) and stored at −20°C until RNA isolation. The RNA was isolated with the RNeasy Kit plus additional Dnase digestion (Qiagen) according to the manufacturer's instructions. The RNA was eluted in 50 µL RNase-free water. The quality of the isolated RNA was checked by gel electrophoresis.
Reverse Transcription.—Samples for real-time PCR were reverse transcribed with the Taqman Reverse Transcription Kit (Applied Biosystems, Foster City, California) according to the manufacturer's protocol. Samples were reverse transcribed with random hexamer primers, and the maximal allowed volume of RNA sample that can be added to the reverse transcription reaction was used.
Primers.—The following primers for CD146 were used: forward primer 5′-CCA AGG CAA CCT CAG CCA TGT C-3′ and reverse primer 5′-CTC GAC TCC ACA GTC TGG GAC GAC T-3′. The resulting amplicon had a size of 437 bp.
Real-Time PCR.—For the amplification of CD146, 1 µL cDNA was added to QuantiTect SYBR Green PCR Master Mix (Qiagen) containing 400 nM forward as well as reverse primers. The PCR was performed using the following thermal settings: 1 cycle of 15 minutes at 95°C and 40 cycles of 15 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C. Relative mRNA expression was calculated with the ▵▵Ct method. As a calibrator the sample with the lowest Ct value was used and set to 100%.
Assessment of FMD on Brachial Artery.—Before the test, the patient was relaxed in a room temperature between 20°C and 25°C; smoking was prohibited. The diameter of brachial artery was measured incident with the R wave of the electrocardiograph trace (Di). Then ischemia was induced by inflating the pneumatic cuff to a pressure 50 mm Hg above systolic, in order to obliterate the brachial artery and induce ischemia. After 5 minutes, the cuff was deflated, and the diameter was measured 60 seconds postdeflation (Df). Flow-mediated dilation was calculated with the formula FMD = [(Df − Di)/Di] × 100.
Statistical Analysis
Data were imported into Statistical Package for the Social Sciences (version 10.0, SPSS, Chicago, Illinois) software for analysis. The χ2 test, paired t test, and correlation test were used to test differences for significance. P value was set at <.05 for significant results.
RESULTS
The clinical characteristics of the studied groups are listed in Table 1, and results of SLE-specific tests and SLE Disease Activity Index are presented in Table 2. There was significant positive correlation between CECs and C3 and anti–double-stranded DNA, and significant negative correlation between CEC and FMD%, disease activity in SLE patients, and total cholesterol, as shown in Table 3.
COMMENT
The patients with SLE have a high incidence of atherosclerosis, with its main consequence, coronary artery disease.23 Anatomopathologic investigations have revealed that the SLE patients were prone to develop premature atherosclerosis.24 The increased risk of atherosclerosis is not exclusively related to traditional risk factors alone.4 Systemic lupus erythematosus itself appeared as an independent risk factor for atherosclerosis, acting through autoimmune vascular injury.8
In patients with systemic lupus erythematosus, atherosclerosis has a long period of subclinical evolution. The first reversible step in the atherogenesis process is represented by endothelial dysfunction.20 Endothelial dysfunction appears when the normal functions of the endothelial cells (control of vascular tone and blood pressure, regulation of leukocyte traffic from the blood to the tissues, platelet adhesion and aggregation, maintenance of the balance between blood coagulation and fibrinolysis, and control of growth, development, and differentiation of the vessel wall cells) are lost or dysregulated.24 Noninvasive methods for the assessment of endothelial dysfunction are represented by flow-mediated vasodilation (endothelium-dependent dilation) and quantification of CECs by flow cytometric analysis.23 Several authors have studied endothelial dysfunction in SLE patients by only FMD, such as Lima et al,25 Tani et al,21 Piper et al,26 and Turner et al.23 In the present study we found a strong inverse correlation between CEC and SLE Disease Activity Index (r = −0.7321, P < .001). This index comprises 24 items (clinical, biological, immunologic): seizure, psychosis, organic brain syndrome, visual disturbance, cranial nerve disorder, lupus headache, cerebrovascular accident, vasculitis, arthritis, myositis, urinary casts, hematuria, proteinuria, pyuria, rash, alopecia, mucosal ulcers, pleurisy, pericarditis, low complement, increased DNA binding, fever, thrombocytopenia, and leukopenia.8 Endothelial dysfunction is caused by several factors, such as anti–double-stranded DNA antibodies (r = 0.72, P < .001), activated complement products (r = 0.71, P < .001), and total cholesterol (r = −0.44, P = .01). Other factors may be involved in this dysfunction, such as systemic inflammation and other antibodies such as antiribonucleoproteins, antiendothelial cells, and antiphospholipids. Clancy et al17 found a tendency toward a correlation between CEC levels and anticardiolipin immunoglobulin G levels in patients with active SLE.
In the SLE group, a significant correlation between the presence of CECs and abnormal FMD% was found by using a nonparametric measure (Spearman r = −0.6321, P < .001). Rajagopalan et al27 reported an inverse correlation between FMD% and the presence of endothelial cell apoptosis in SLE and concluded that apoptotic CECs (CD146+ annexin V+) measured by flow cytometric analysis were the only variable that predicted FMD in women with SLE.
In the present study CD146 was assessed by real-time PCR, which offers several advantages. It is possible to freeze the blood samples, facilitating the integration of such measurement in routine clinical work, and standardization of the real-time PCR method is easier than standardization of flow cytometric analysis. Further, in flow cytometry there is always the danger of unspecific staining with antibodies, especially in patients with activated leukocytes, and the detection of platelet aggregates as cells. This could in particular influence the detection of such tiny populations as the CECs.28
CONCLUSION
Endothelial dysfunction is present in SLE patients even in the absence of traditional cardiovascular risk factors. It is due to disease activity. Measurement of CECs using CD146 by real-time PCR represents a noninvasive method for the assessment of endothelial dysfunction in this study.
