Rheumatol Int DOI 10.1007/s00296-008-0676-7
O R I G I N A L A R T I CL E
Normal levels and function of endothelial progenitor cells in patients with psoriatic arthritis Jacob N. Ablin · Zacharinka Goldstein · Valerie Aloush · Hagit Matz · Ori Elkayam · Dan Caspi · Shmuel Swartzenberg · Jacob George · Yonit Wohl
Received: 9 February 2008 / Accepted: 1 August 2008 © Springer-Verlag 2008
Abstract Endothelial progenitor cells (EPCs) are a population of bone marrow derived cells which have been attributed with the ability to migrate into areas of tissue ischemia and to posses reparative qualities. EPCs have been shown to be decreased in level and function in various inXammatory disorders. Psoriasis and psoriatic arthritis are associated with an increase in cardiovascular morbidity. The aim of the study was to investigate the number of EPCs among patients suVering from psoriasis and psoriatic arthritis. Patients suVering from active psoriasis and psoriatic arthritis were recruited as well as healthy controls. Disease activity was assessed with the DAS-28, BASDAI and PASI scores. Peripheral blood mononuclear cells were isolated and EPC numbers evaluated by FACS analysis using the CD34/133 and CD34/KDR. No signiWcant diVerence was found between numbers of EPCs between healthy controls, patients with psoriasis and psoriatic arthritis. A signiWcant correlation was found between levels of VGEF and the BASDAI score. The results of the current study do not
J. N. Ablin (&) · D. Caspi The Department of Rheumatology, Rheumatology Institute, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel e-mail:
[email protected];
[email protected] Z. Goldstein · V. Aloush · O. Elkayam Department of Internal Medicine F, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel H. Matz · Y. Wohl Department of Dermatology, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel S. Swartzenberg · J. George Cardiology Institute, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel
support a signiWcant role for EPCs in the pathogenesis of psoriasis and psoriatic arthritis. Keywords Psoriatic arthritis · Psoriasis · Endothelial progenitor cells (EPCs) · VGEF
Background Endothelial progenitor cells (EPCs) are a population of bone marrow derived cells characterized by expression of CD34 and VGEFR-2. These cells, which are estimated to constitute about 0.01% of peripheral mononuclear cells, posses the capacity to proliferate, migrate and diVerentiate into mature endothelial cells. Characterized by their capacity to transform into mature endothelial cells, EPCs participate in ongoing endothelial repair, while an imbalance between endothelial damage and repair may lead to an increase in cardiovascular events [1]. EPCs are mobilized from bone marrow and migrate to areas of ischemia and or damaged endothelium, participating in angioneogenesis. Thus, EPCs may possess a reparative function, involved in overcoming vascular damage and reducing cardiovascular risk. Levels of EPCs have been shown to increase in conditions such as unstable angina compared to stable angina [2] and decrease in patients undergoing in-stent restenosis compared to those not undergoing such injury, emphasizing the potentially protective eVect of these cells [3]. Decreased levels of EPCs have been demonstrated among rheumatoid arthritis patients [4], another systemic inXammatory disorder associated with increased cardiovascular morbidity and mortality; we have previously demonstrated an increase in EPC levels following treatment with the anti-TNF agent inXiximab in rheumatoid arthritis [5].
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Rheumatol Int
Psoriasis is a chronic dermatological disorder characterized by epidermal cell proliferation and hyperkeratosis, inWltration of the skin by inXammatory cells, neoangiogenesis, thickening of skin and scaling [6]. Neoangiogenesis is an early stage in the pathogenesis of psoriasis and endothelial cells play an important role in development of the psoriatic plaque. TNF is a central pro-inXammatory cytokine in the pathogenesis of psoriasis and of psoriatic arthritis. It is secreted in large amounts by activated T cells in psoriatic lesions resulting in chronic inXammation, tissue damage and hyperplasia [7]. TNF induces secretion of VGEF, a major regulator of angiogenesis [7, 8]. Thus, anti TNF treatment, e.g. etanercept, possesses the ability to decrease angiogenesis in psoriatic lesions in concordance with the improvement in patients’ clinical condition [8, 9]. Endothelial progenitor cell level and activity is considered as an indicator both of levels of inXammation, levels of vascular damage as well as reXecting reparative potential [10–12]. Various techniques have been described for evaluation and measurement of EPCs. These cells can be isolated in cell culture and their levels inferred through their ability to form characteristic colonies. This technique has been utilized in a series of studies in which EPC levels were evaluated in various pathological conditions [1–3]. On the other hand, multiple studies have made use of FACS sorting to identify and characterize EPCs, based on the expression of speciWc cell surface makers including CD133, CD34, KDR and VGEF-receptor-2 [4, 13–16]. This method, which has been used in the current study, has signiWcant advantages regarding accuracy. The level and function of EPCs have not been previously evaluated in psoriatic arthritis. This information has potential signiWcance regarding the pathogenesis of both psoriasis and psoriatic arthritis and could indicate a novel therapeutic avenue for these disorders. Thus, the objective of the current study was to evaluate the levels and function of EPCs among patients suVering from psoriatic arthritis while assessing the correlation between these levels and both clinical parameters of disease activity as well as standard measures of inXammation such as CRP as well as levels of VEGF, a central regulator of angiogenesis.
Methods The study was conducted after approval of the institutional Helsinki committee and written informed consent was obtained from all participants. Psoriatic arthritis patients were recruited among patients attending a referral rheumatology clinic. Patients with either symmetrical peripheral involvement, asymmetrical peripheral involvement or axial involvement were recruited. Healthy normal controls were
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recruited among the hospital staV. Exclusion criteria included known cardiovascular disorders, including ischemic heart disease or prior event of CVA, diabetes and active malignancy. Demographic data were recorded as well as information regarding medication use, including anti-inXammatory agents (e.g. NSAIDS), statins etc. Use of anti TNF medications was documented. Clinical assessment Patients underwent clinical evaluation at time of recruitment. Dermatological involvement was documented using the PASI score and articular involvement was documented using a complete 66 joint examination. Axial involvement was evaluated by use of the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) [17], Schober test, lateral spinal Xexion, chest expansion and cervical rotation. Dactyilitis was documented. Based on the clinical characteristics, patients were divided into groups with high and low disease activity. This distinction, which was subsequently utilized for analysis of results, was performed separately based on each clinical parameter. Thus, patients were divided based on the DAS28 to groups with scores above and below a value of 3.2. Based on the BASDAI patients were divided into groups with scores above and below a value of 3.5. Similarly patients were divided based on the PASI results into groups with values above and below a value of 5. EPC isolation Blood samples were acquired upon recruitment from all patients. Mononuclear cells were isolated by Ficoll density gradient centrifugation and separate samples were retained for subsequent evaluation of levels of CRP and VEGEF. A total of 1.5 £ 106 cells were taken for FACS analysis. FACS analysis was performed with utilization of the following three markers: 1. Phycoerythrin anti-CD34 (IQ products) 2. Allophycocyanin anti-VEGF receptor-2 (KDR, R&D Systems, Minneapolis, MN) 3. FITC-anti CD 133 (R&D systems). Cells were incubated with the marker for 30 min in the presence of 100 L Xuorescence-activated cell sorter staining buVer (FACS buVer) (phosphate-buVered saline and 2% fetal calf serum) at 4°C. Subsequently cells were washed with phosphate buVered saline (PBS) and Wxed with formaldehyde for up to 24 h prior to FACS reading. FACS analysis was performed with use of Calibur; Becton Dickinson equipment and CellQuest software (BD Bioscience). A total of 80,000 cells were counted at each FACS reading (Fig. 1).
Rheumatol Int Table 1 Clinical parameters of patients in three study groups Characteristic
Group 1: controls (n = 16)
Group 2: skin psoriasis (n = 10)
Group 3: psoriatic arthritis (n = 22)
Demographics
41.69 § 9.71
48.6 § 18.6
47.18 § 8.15
Male n (%)
4 (25)
6 (60)
12 (54.5)
Current smoking n (%)
7 (43)
7 (70)
3 (13.6)
Hypertension
0 (0)
1 (10)
8 (36.4)
Hyperlipidemia
0 (0)
1 (10)
3 (13.6)
Age (years) § SD
Medical history n (%)
Clinical parameters Systemic therapy n (%)
Fig. 1 FACS analysis of CD34 cells
Evaluation of VGEF and CRP levels Levels of VGEF were determined by use of a commercial ELISA kit (Biosource Camarillo, CA). High sensitivity CRP was determined by ELISA.
Statistics Comparison between the groups with regard to demographic (gender, age), risk factors (diabetes mellitus, hypertension, hyperlipidemia, smoking, etc.) and clinical factors (EPC numbers, VGEF, CRP etc.), was performed using unpaired Student’s t tests, Mann–Whitney and Chi-square tests, as applicable. ANOVA was performed for each parameter as described in the results. Spearman’s correlation coeYcients were calculated in the groups to study the relationship between CD34/133 and CD34/KDR cells and disease activity. The statistical signiWcance level was set to 0.05 and the SPSS (Chicago, IL) for Windows software, Version 13.0 was used for the analysis.
Results Table 1 presents demographic data regarding patients in the three groups: group 1—healthy controls (n = 16); group 2—psoriatic patients with no articular involvement (n = 10) and group 3—psoriatic arthritis patients (n = 22). As is evident from the data presented, the groups did not signiWcantly diVer regarding basic demographic characteristics (age, gender, BMI and background medical history).
Anti-TNF
0 (0)
0 (0)
12 (54.5)
MTX
0 (0)
0 (0)
7 (31.8)
ACE inhibitors
0 (0)
1 (10)
2 (9.1)
Statins
0 (0)
1 (10)
3 (13.6)
Aspirin
0 (0)
0 (0)
1 (4.5)
NSAID’s
0 (0)
0 (0)
6 (27.3)
24.4 § 3.63
27.05 § 7.09
27.70 § 5.34
BMI
It must be pointed out that the proportion of smokers was signiWcantly higher among controls and psoriatic patients (groups 1 and 2) compared with group 3 (psoriatic arthritis patients). It is also noteworthy that patients treated with anti-TNF were found only in group 3. Evaluation of EPC levels among the 3 groups Table 2 presents the proportion of cells which stained positive for the abovementioned FACS markers. These cells represent the population of EPCs. Statistical analysis of these results, using ANOVA calculation, shows the absence of any signiWcant diVerence between the three groups. As noted above, in addition to comparing the groups with regard to levels of EPCs, comparison was performed regarding levels of VGEF and CRP. Table 3 presents levels of these two parameters between study groups. Statistical analysis of these results by use of ANOVA demonstrated no signiWcant diVerence except for higher levels of CRP among patients with psoriatic arthritis compared to groups 1 and 2 (as expected). To further characterize the possibility of a diVerence between groups diVering on clinical grounds, further analysis was carried out by subdividing patients into groups with high and low disease activity based on the scores obtained on the DAS-28 scale, using a cutoV of 3.2, based on the BASDAI—using a cutoV of 3.5 and based on the PASI using a cutoV of 5. These groups were once again compared regarding levels of EPCs, VGEF and CRP. The results of
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Rheumatol Int Table 2 Mean levels of CD34/133 positive and CD34/KDR positive cells among patients from the three study groups N
Mean
SD
95% CI for mean Lower bound
Minimum
Maximum
Upper bound
CD34/133 Control
15
0.035
0.035
0.016
0.055
0.000
0.110
Psoriasis
9
0.038
0.049
0.000
0.075
0.010
0.160
Psoriasis + arthritis
22
0.022
0.025
0.011
0.033
0.000
0.100
Total
46
0.029
0.034
0.019
0.039
0.000
0.160
CD34/KDR Control
15
0.035
0.031
0.018
0.052
0.000
0.090
Psoriasis
9
0.019
0.019
0.004
0.033
0.000
0.060
Psoriasis + arthritis
22
0.025
0.021
0.016
0.035
0.000
0.100
Total
46
0.027
0.025
0.020
0.034
0.000
0.100
Minimum
Maximum
Table 3 Levels of VGEF and CRP between study groups N
Mean
SD
95% CI for mean Lower bound
Upper bound
VEGF Control
15
363.66
278.91
209.21
518.12
28.3
Psoriasis
10
509.37
357.88
253.36
765.37
105.2
1080.3 1188.6
Psoriatic arthritis
22
409.03
313.22
270.15
547.90
55.1
1420.4
Total
47
415.90
310.38
324.77
507.03
28.3
1420.4
Control
15
1.84
3.03
0.17
3.52
0.08
11.42
Psoriasis
10
5.71
8.05
¡0.05
11.47
0.03
26.71
Psoriatic arthritis
22
15.73
18.61
7.48
23.98
0.30
80.39
Total
47
9.16
14.64
4.87
13.46
0.03
80.39
CRP
these analyses demonstrated no signiWcant diVerence in the levels of EPCs, or VGEF among patients with high disease activity and patients with low disease activity based on the abovementioned parameters, although a non-signiWcant trend appeared to exist towards a decrease in the levels of CD34/133 positive cells among patients with high disease activity (high DAS-28 scores). The only (expectable) diVerence noted was a signiWcantly higher level of CRP among patients with high disease activity, verifying the validity of the clinical classiWcation. To further evaluate the possibility of a relationship between EPC levels and the clinical data collected for our patient population, Pearson correlation coeYcient was calculated for the relationship between the DAS-28, the BASDAI and the PASI and between the levels of CD34/CD133 positive cells, CD34/KDR positive cells as well as the levels of VGEF and CRP. The results, presented in Table 4, showed a signiWcant correlation between BASDAI values and levels of VGEF. PASI levels were signiWcantly correlated with CRP levels. No signiWcant correlations were
123
identiWed between any clinical index and the levels of CD34/133 or CD34/KDR cells.
Discussion In the current study, no signiWcant diVerence was found between EPC levels among healthy controls, patients with psoriasis and patients with psoriatic arthritis. As previously mentioned, a number studies have previously demonstrated the association between EPC levels and/or function and between various forms of inXammation as well as conditions associated with tissue ischemia and cardiovascular morbidity. In addition psoriasis, in its dermatological manifestations, is associated with endothelial proliferation and has been linked with complex alterations in the levels of various inXammatory and angiogenic mediators including VGEF. This protein, which constitutes a major angiogenic regulator, is over expressed in the skin of psoriatic patients [6, 7, 18]. EPCs, characterized by their
Rheumatol Int Table 4 Pearson correlation between clinical indices (DAS-28, BASDAI, PASI) and levels of CD34/133 and CD34/KDR CD34_133
CD34_KDR
VEGF
CRP
0.547**
PASI Pearson correlation
0.070
0.322
0.218
Sig. (2-tailed)
0.707
0.077
0.232
0.001
N
31
31
32
32
Pearson correlation
¡0.153
¡0.333
0.427*
0.316
Sig. (2-tailed)
0.495
0.130
0.048
0.152
N
22
22
22
22
Pearson correlation
¡0.242
¡0.302
0.411
0.338
Sig. (2-tailed)
0.290
0.183
0.064
0.134
N
21
21
21
21
BASDAI
DAS28
* Correlation is signiWcant at the 0.05 level (2-tailed) ** Correlation is signiWcant at the 0.01 level (2-tailed)
ability to create new blood vessels, have been previously studied among patients with rheumatoid arthritis [4, 5] and additional inXammatory disorders such as inXammatory bowel disease [16]. A number of studies have demonstrated defects in the levels or function of EPCs among patients suVering from these disorders, patients who are classically considered to be at an increased risk regarding cardiovascular morbidity and mortality [19–21]. In view of these results, the assumption has been made that defective EPC function may play a role in the increased cardiovascular morbidity and mortality in these conditions. In this venue it is noteworthy that in rheumatoid arthritis anti-inXammatory treatment can improve the levels and function of EPCs [5] in association with the clinical improvement; anti-inXammatory treatment has also been shown to decrease the cardiovascular risk associated with inXammatory arthritis [19, 22, 23]. In view of this background, in the current study we attempted to test the hypothesis that the levels of EPCs may be impaired among patients with psoriasis and psoriatic arthritis as well. Such a defect would be a possible explanation for the increase in cardiovascular morbidity which is observed in this patient population [19, 24–26]. While evaluating the negative results of the current study a number of points are worthy of notice. First, as is always the case in the event of negative results, the possibility exists that increasing the size of the sample may have exposed more subtle diVerences between the groups. The trend towards decreased levels of EPCs among patients with high DAS28 scores may hint towards this possibility. Another confounding factor is related to the demographic characteristics of the control group which included a higher proportion of smokers thus potentially obscuring diVerences in EPC function between the groups.
On the theoretical levels, it is possible that the increased levels of VGEF (at least at the level of the skin) observed among psoriasis patients may have a balancing eVect regarding EPC levels in contrast to other inXammatory disorders, thus resulting in a EPC level within the normal range (although not necessarily of normal function). Another point to mention is that the patient population examined was heterogeneous with regard to medications. Some of the patients were treated with anti-TNF medications, known to posses the ability to increase EPC levels and improve their function [27]. This heterogeneity may have further acted as a confounding factor. Lately some disagreement has arisen regarding the deWnition and identifying characteristics of EPCs. These cells were previously identiWed by their ability to proliferate in cell culture and create clusters with a typical morphology [1], while subsequently FACS has been more commonly used to identify these cells by typical cell surface markers. Unfortunately in many cases a lack of correlation was found between the cells identiWed by diVerent techniques [14, 28]. Recently the claim has been made that the “real” EPC population is diVerent from the cells previously studied, a population described as endothelial colony forming cells (ECFC) [29]. This theoretical lack of clarity causes diYculty in interpretation of EPC studies as in the current case.
Conclusion In the current study we have attempted to characterize the levels of EPCs among patients suVering from psoriasis and psoriatic arthritis using FACS analysis for the identiWcation of the characteristic cell surface markers CD34/133 and CD34/KDR. In the current study no signiWcant diVerence was identiWed between the level of these cells among patients compared to controls and no signiWcant diVerence was found between patients with high or low disease activity based on accepted clinical indices. A positive correlation was found between the BASDAI score and the level of VGEF, a central mediator of angiogenesis. These results do not support a role for EPCs in the pathogenesis of cardiovascular morbidity among psoriatic patients.
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