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Effect of anti-TNFα treatment on circulating endothelial progenitor cells (EPCs) in rheumatoid arthritis
Life Sciences 79 (2006) 2364 – 2369 www.elsevier.com/locate/lifescie
Effect of anti-TNFα treatment on circulating endothelial progenitor cells (EPCs) in rheumatoid arthritis Jacob N. Ablin a,⁎, Viktoria Boguslavski b , Valerie Aloush a , Ori Elkayam a , Daphna Paran a , Dan Caspi a , Jacob George c b
a Department of Rheumatology, Tel-Aviv Sourasky Medical Center and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel Department of Internal Medicine 6, Tel-Aviv Sourasky Medical Center and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel c Department of Cardiology, Tel-Aviv Sourasky Medical Center and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
Received 11 July 2006; accepted 31 July 2006
Abstract Abstract: Rheumatoid arthritis (RA) is associated with increased cardiovascular morbidity and mortality, which may be attenuated by anti-inflammatory treatment. Endothelial progenitor cells (EPCs) have the ability to differentiate into mature endothelium and have a potentially reparative role protecting against ischemia and atherosclerosis. Objective: To investigate the effect of treatment with infliximab on the number and functional capacity of endothelial progenitor cells (EPCs) in patients with RA, as a possible mechanism for reducing cardiovascular morbidity in this disorder. Methods: Patients: Active seropositive RA patients (N = 14) considered candidates for starting infliximab treatment, were recruited. Assessment, based on DAS-28, was performed before treatment and 14 days later. Peripheral blood mononuclear cells were isolated and EPC numbers evaluated by the colony-forming unit (CFU) method. Endothelial phenotyping of CFU was performed by immunofluorescence employing antibodies to Tie-2 VEGF-receptor 2, and CD31. EPC Functional properties were evaluated by fibronectin adherance. Results: A significant 33.4% increase (p b 0.001) in EPC levels was observed after infliximab. A 60% increase was noted in the EPC differentiation scale, (p b 0.002) while a 37.6% increase was observed in mean EPC adhesion (p b 0.001). These changes were associated with a 17.5% decrease in the DAS-28 (p b 0.0001). A significant correlation was observed between the clinical response, reflected by changes in DAS-28 and the degree of increase in EPC CFUs. Conclusion: A single dose of infliximab improved the number and functional properties of EPCs, in parallel with an early clinical effect, suggesting a possible mechanism by which anti-inflammatory treatment may reduce cardiovascular risk in RA patients. © 2006 Elsevier Inc. All rights reserved. Keywords: Rheumatoid arthritis; Anti-TNFα; EPC; Angiogenesis
Introduction Increased cardiovascular mortality in rheumatoid arthritis (RA) is well established (Wolfe et al., 1994) though recent evidence indicates it may be declining, possibly as a result of earlier and tighter control of inflammation (Krishnan et al., 2004). Systemic inflammation is associated with worsened cardiovascular outcome (Mulvihill and Foley, 2002) and the presence of an inflammatory component plays an active role in the progression of the atherosclerotic plaque. This has led to ⁎ Corresponding author. Tel.: +972 3 6973668; fax: +972 3 6916163. E-mail address: [email protected] (J.N. Ablin). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.07.035
evolution of the concept of atherosclerosis as an extra-articular manifestation of RA (Van Doornum et al., 2002). Moreover, it has been established that effective suppression of disease activity by methotrexate can reduce mortality of rheumatoid arthritis patients (Choi et al., 2002). Endothelial progenitor cells (EPCs) are a recently identified population of circulating bone marrow-derived cells characterized by their ability to migrate into areas of increased angiogenesis and differentiate into mature endothelial cells (Asahara et al., 1997; Murohara et al., 2000). EPCs are regarded as a population with reparative capacities which have the ability to ameliorate vascular injury, thereby combating conditions of compromised tissue perfusion. The transfer of EPCs is under evaluation as a therapeutic
J.N. Ablin et al. / Life Sciences 79 (2006) 2364–2369 Table 1 Clinical characteristics of study patients Mean age (SD) Gender male/female Mean BMI (SD) Rheumatoid factor positive DAS-28 (mean, SD) Medications Hydroxychloroquine Sulfasalazine Methotrexate (average dose) Prednisone (average dose)
55.1 (11.36) 3 / 11 24.5 (3.0) 100% 5.1 (1.4) 1 / 14 (7.1%) 1 / 14 (7.1%) 10 / 14 (71.4%) (12.5 mg/week) 11 / 14 (78.6%) (6.8 mg/d)
modality potentially effective both in ischemia-related conditions such as myocardial infarction (Kawamoto et al., 2001) and in nonischemic inflammatory models such as dilated cardiomyopathy (Werner et al., 2005). EPC levels have been shown to be negatively correlated with risk factors for atherosclerosis (Hill et al., 2003), suggesting that decreased repair of vascular plaque may be the result of EPC depletion. EPCs have been identified in synovial tissue of RA patients where they may participate in angiogenesis (Ruger et al., 2004). On the other hand, it has recently been demonstrated that EPCs are depleted in the peripheral blood of RA patients (Grisar et al., 2005) and this finding has been interpreted as a possible contributing factor to the increased cardiovascular morbidity and mortality of patients suffering from this disease. Interestingly, treatment with recombinant human erythropoietin, which has been shown to improve both anemia and clinical indices of rheumatoid arthritis (Peeters et al., 1999), has recently been demonstrated to promote EPC proliferative and adhesive properties (George et al., 2005). TNFα is a central pro-inflammatory cytokine involved in pathogenesis of both RA (Choy and Panayi, 2001) and atherosclerosis (Young et al., 2002). Infliximab, a chimeric antibody targeted against TNFα, has become a mainstay of treatment in RA over the last years (Lipsky et al., 2000). Infliximab has been shown to improve endothelial function in patients with RA (Hurlimann et al., 2002; Gonzalez-Juanatey et al., 2004). Recent research implies this effect is irrespective of lipid profile (Vis et al., 2005). In the current study we have attempted to determine the effect of infliximab treatment on the number and function of EPCs in RA patients as a possible mechanism for the effect of this treatment on endothelial function. Patients and methods Study subjects Fourteen consecutive RA patients who fulfilled the American College of Rheumatology (ACR) criteria for classification of RA (Arnett et al., 1988) were recruited from the tertiary care rheumatology clinic of the Tel-Aviv Sourasky Medical Center. All participating patients had been considered eligible for infliximab treatment based on clinical assessment by the attending rheumatologist or were already being treated with infliximab. The standard treatment protocol implemented in these patients con-
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sisted of an infusion of infliximab 3 mg/kg on weeks 0, 2, 6 and subsequently every 8 weeks. Exclusion criteria included diabetes mellitus or a history of coronary artery disease, cerebrovascular disease or intermittent claudication. These patients were excluded in order to limit confounding effects on endothelial function. Although not originally defined as an inclusion criterion, all patients were sero-positive for Rheumatoid Factor (RF). This study was approved by the institutional review board and was carried out in accordance with the ethical principles of the declaration of Helsinki. All patients underwent clinical and laboratory assessment at the time of recruitment, which timed on the day of infliximab treatment and 2 weeks later. This included physical examination and a standard 28 joint count as the basis for determining disease activity 28 (DAS-28) score (Prevoo et al., 1995) and laboratory assessment of clinical activity (ESR, CRP). Subsequently, blood samples were obtained for EPC isolation before starting infliximab infusion. Isolation of EPCs and colony-forming unit assay Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density-gradient centrifugation (Sigma) from 20-ml blood samples and EPC CFU was assayed after two platings and a 9-day culture on fibronectin-coated, 24-well plates, as described (George et al., 2003; Hill et al., 2003). Colonies were manually counted in a minimum of 3 wells by two independent observers who were unaware of the patients' clinical data. Results were expressed as CFU/well in four separate wells from the same patient. Confirmation of CFU phenotype For phenotyping of endothelial characteristics of colonyforming unit (CFU), the following antibodies were used in immunofluorescence and flow cytometric analysis: rabbit polyclonal anti-Tie-2 (C-20), mouse monoclonal anti-flk-1(A-3), and goat polyclonal anti-CD31 (PECAM-1, M-20), all from Santa Cruz. Endothelial cell lineage was further confirmed by indirect immunostaining with the use of 1,1-dioctadecyl3,3,3,3-tetramethylindocarbocyanine perchlorate-acetylated low-density lipoprotein (DiI-acLDL) and co-staining with BS1 lectin (Sigma). Fibronectin adhesion assay EPCs (day 7) were washed with PBS and gently detached with 0.5 mmol/l EDTA in PBS. After centrifugation and resuspension in basal complete medium supplemented with 5% FCS, identical cell numbers were placed on fibronectin-coated culture dishes and incubated for 30 min at 37 °C (Tepper et al., 2002; George et al., 2003). Adherent cells were counted by independent blinded investigators. Results Demographic and clinical characteristics of the patients are presented in Table 1. As noted, all patients were RF positive. 10 out
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Fig. 1. A: EPC CFUs before and 14 days after treatment with infliximab. A significant increase in number of EPC CFUs, determined as described in Patients and methods, is demonstrated 14 days after treatment with infliximab. B: EPC adhesion before and 14 days after treatment with infliximab. A significant increase is observed in EPC adhesion as measured by adherence to fibronectin (see Patients and methods), 14 days after treatment with infliximab. C: Effect of infliximab treatment on EPC differentiation. EPC differentiation was measured by two separate observers and scored on a semi-quantitative scale. A significant increase in EPC differentiation is observed after treatment with infliximab. D: Reduction of DAS-28 after infliximab treatment. Clinical assessment of patients was documented by determination of the DAS-28 score. The figure demonstrates the clinical response to infliximab reflected by a significant reduction of the DAS-28 14 days after treatment with infliximab.
J.N. Ablin et al. / Life Sciences 79 (2006) 2364–2369 Table 2 EPC colonies before, and 2 weeks after treatment with infliximab Patient number
Colonies before treatment
Colonies after treatment
Δ (absolute)
Δ (%)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
25 31 97 24 45 47 30 69 57 32 18 99 20 58
32 59 102 52 42 75 64 75 52 48 32 120 55 62
7 28 5 28 −3 28 34 6 −5 16 14 21 35 4
28 90.3 5.6 117 − 6.7 59.6 113 8.7 − 8.8 50 77.8 21.2 175 6.9
of 14 patients (71.4%) were treated with methotrexate while 11 / 14 (78.6%) were on corticosteroids. The average duration of prior treatment with infliximab was 9.6 months (range — 1–32 months, SD — 26.2). A significant 33.4% increase [from 46.6(SD — 26.7) to 62.1(SD — 24.7) CFU/well; pb 0.001] in EPC levels was observed 14 days after infliximab treatment (Fig. 1A). Table 2 presents the absolute and percentage change in EPC CFUs following treatment with infliximab. Aiming to determine whether infliximab treatment influences the functional properties of EPC, we performed adhesion assays. A 37.6% increase was observed in mean number of adhering EPC from 75.43 (SD — 41.0) to 103.79 (SD — 42.5) cells per plate ( pb 0.001) (Fig. 1B). A 60% increase was noted in the EPC differentiation scale, from 2.5 (SD — 1.6) to 4.0 (SD — 1.1) ( pb 0.002) (Fig. 1C). These changes were associated with a 17.5% decrease in the DAS-28 from 5.1 (SD — 1.4) to 4.2 (SD — 1.1) ( p b 0.0001) (Fig. 1D), reflecting the clinical response to infliximab. A significant correlation was observed between the change in DAS-28 and the change in EPC CFU (Fig. 2) implying an interrelation between this clinical index and the physiologic effect of anti-TNFα treatment on EPCs.
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Herbrig et al. have shown a clear correlation between endothelial dysfunction in RA patients and impaired function of EPCs (Herbrig et al., 2005). Depletion of EPCs in RA has previously been shown by Grisar et al. who studied 52 patients at different levels of disease activity and treated with a variety of DMARDs. They observed an inverse correlation between the number of EPCs and disease activity. EPC levels were not affected by the use of DMARDs except for anti-TNFα therapy, which, in 4 patients, was associated with a higher number of EPCs, similar to healthy controls (Grisar et al., 2005). Our study confirms and further substantiates this observation, demonstrating the dynamic effect of infliximab on both the number and function of EPCs, in consistence with the favorable effect of infliximab on endothelial function of RA patients (Hurlimann et al., 2002; Gonzalez-Juanatey et al., 2004). Our results demonstrate the capacity of anti-TNFα treatment to improve EPC levels and function in parallel with their clinical anti-inflammatory effect. Since EPCs are considered reparative cells, capable of ameliorating ischemic damage (Takahashi et al., 1999), this finding implies a potential mechanism through which this potent anti-inflammatory treatment may act to decrease the vasculopathy involved in RA (and possibly other chronic inflammatory conditions) and which may translate into reduced cardiovascular morbidity and mortality. Our results offer a novel viewpoint on the effect of anti-inflammatory treatment on vascular biology in rheumatoid arthritis. Although the link between this prototypical inflammatory joint disease and both atherosclerosis and cardiovascular morbidity and mortality has been epidemiologically well established, the role of endothelial progenitor cells, and the effect of anti-inflammatory treatment on this population have not been previously studied. Hence, the improved EPC levels and function observed here could provide a plausible mechanistic explanation. Moreover, these results could point towards the potential to develop novel therapeutic interventions in rheumatoid arthritis and in similar inflammatory disorders which would specifically target mediators which down-regulate EPCs. This possibility is highlighted by the recent results demonstrating that TNFα can directly inhibit EPCs (Seeger et al., 2005). An alternative approach would be to pursue medications which, similar to HMG-CoA-reductase inhibitors
Discussion In this study, we have shown that a single dose of infliximab treatment improved the number and functional properties of EPCs in patients with RA, in parallel with the expected clinical improvement. A significant correlation was observed between the extent of clinical improvement (reflected by the DAS-28) and the level of increase in the number and function of EPC CFUs. This clear effect suggests a potential novel mechanism which may explain the salutory effect of anti-inflammatory treatment on the cardiovascular risk associated with rheumatoid arthritis. Rheumatoid arthritis is characterized by impaired endothelial function (Vaudo et al., 2004; Herbrig et al., 2005). Young to middle aged patients with RAwith low disease activity, free from cardiovascular risk factors and cardiovascular disease, have altered endothelial reactivity, probably related to the underlying inflammatory condition (Vaudo et al., 2004). Furthermore,
Fig 2. Correlation between improvement in DAS-28 and change in EPC CFUs. Patients with moderate to severe RA were evaluated for their DAS-28 scores and EPC numbers prior to and after a course of infliximab. A correlation was sought between EPC numbers and DAS-28 as described in Patients and methods.
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(Assmus et al., 2003) and erythropoietin (George et al., 2005), directly stimulate EPCs. Notably, EPCs play additional roles in rheumatoid arthritis. Ruger et al have demonstrated the presence of endothelial progenitor cell in synovial tissue of RA patients (as well as osteoarthritis) suggesting a role for these cells in the neovascoulogenesis involved in the formation of panus (Ruger et al., 2004). Thus, while EPCs are presumed to be systemically protective against ischemia, they may simultaneously facilitate the proliferative process responsible for cartilage destruction in RA. Moreover, TNFα may play a complex role in this cascade. Thus, while TNFα has a pro-apoptotic effect on bone marrow-derived progenitor cells (thus contributing to inflammatory induced anemia) it appears to protect inflammatory cells within the RA joint (e.g. macrophages and synovial fibroblasts), thus contributing to joint destruction in RA (Liu and Pope, 2003). TNFα also appears to down-regulate EPCs through additional pathways not dependent on apoptosis (Seeger et al., 2005). Since the measured serum levels of EPCs may represent the product of a balance between bone marrow production and mobilization and peripheral apoptosis and breakdown, a logical subsequent step after the current study would be to try and elucidate the correlation between levels of bioavailable TNFα and the effect on EPCs. Determining such levels would however be particularly difficult due to the binding of TNFα by infliximab, a specific monoclonal antibody aimed against that cytokine. In our study, we employed the CFU method for assessment of EPC numbers. This method employs evaluation of formation of clusters of colonies consisting of a core of concentrated cells with eccentrically ‘protruding’ spindle shaped cells. These colonies are routinely stained for several endothelial cell markers such as Tie-1, KDR and CD31 and the results are also crossvalidated by double staining for a lectin (BS-1) and a receptor for acetylated LDL. We are aware of the current debate as to the accurate methodology with regard to assessment of EPC num bers. However, neither consensus nor standardization has hitherto been achieved and several groups are employing the CFU method (Hill et al., 2003; George et al., 2003; Assmus et al., 2003), whereas others favor analysis of FACS markers that are equally problematic (some regard EPC as CD34+CD133+, others CD34+/KDR and yet others prefer the CD133/KDR). In a very recent study (George et al., 2006), we have compared all these methods and found that they did not correlate, yet CFUs were correlated with functional properties consistent with proliferation of endothelial like cells. In conclusion, we have found that infliximab enhances the number and function of EPCs in patients with moderate to severe rheumatoid arthritis in correlation with the clinical improvement. This finding may be suggestive of a novel mechanism by which anti-inflammatory treatment in general and anti-TNFα treatment in particular may alter the increased cardiovascular morbidity and mortality associated with rheumatoid arthritis. References Arnett, F.C., Edworthy, S.M., Bloch, D.A., McShane, D.J., Fries, J.F., Cooper, N.S., Healey, L.A., Kaplan, S.R., Liang, M.H., Luthra, H.S., et al., 1988. The American
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Effect of anti-TNFα treatment on circulating endothelial progenitor cells (EPCs) in rheumatoid arthritis Jacob N. Ablin a,⁎, Viktoria Boguslavski b , Valerie Aloush a , Ori Elkayam a , Daphna Paran a , Dan Caspi a , Jacob George c b
a Department of Rheumatology, Tel-Aviv Sourasky Medical Center and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel Department of Internal Medicine 6, Tel-Aviv Sourasky Medical Center and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel c Department of Cardiology, Tel-Aviv Sourasky Medical Center and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
Received 11 July 2006; accepted 31 July 2006
Abstract Abstract: Rheumatoid arthritis (RA) is associated with increased cardiovascular morbidity and mortality, which may be attenuated by anti-inflammatory treatment. Endothelial progenitor cells (EPCs) have the ability to differentiate into mature endothelium and have a potentially reparative role protecting against ischemia and atherosclerosis. Objective: To investigate the effect of treatment with infliximab on the number and functional capacity of endothelial progenitor cells (EPCs) in patients with RA, as a possible mechanism for reducing cardiovascular morbidity in this disorder. Methods: Patients: Active seropositive RA patients (N = 14) considered candidates for starting infliximab treatment, were recruited. Assessment, based on DAS-28, was performed before treatment and 14 days later. Peripheral blood mononuclear cells were isolated and EPC numbers evaluated by the colony-forming unit (CFU) method. Endothelial phenotyping of CFU was performed by immunofluorescence employing antibodies to Tie-2 VEGF-receptor 2, and CD31. EPC Functional properties were evaluated by fibronectin adherance. Results: A significant 33.4% increase (p b 0.001) in EPC levels was observed after infliximab. A 60% increase was noted in the EPC differentiation scale, (p b 0.002) while a 37.6% increase was observed in mean EPC adhesion (p b 0.001). These changes were associated with a 17.5% decrease in the DAS-28 (p b 0.0001). A significant correlation was observed between the clinical response, reflected by changes in DAS-28 and the degree of increase in EPC CFUs. Conclusion: A single dose of infliximab improved the number and functional properties of EPCs, in parallel with an early clinical effect, suggesting a possible mechanism by which anti-inflammatory treatment may reduce cardiovascular risk in RA patients. © 2006 Elsevier Inc. All rights reserved. Keywords: Rheumatoid arthritis; Anti-TNFα; EPC; Angiogenesis
Introduction Increased cardiovascular mortality in rheumatoid arthritis (RA) is well established (Wolfe et al., 1994) though recent evidence indicates it may be declining, possibly as a result of earlier and tighter control of inflammation (Krishnan et al., 2004). Systemic inflammation is associated with worsened cardiovascular outcome (Mulvihill and Foley, 2002) and the presence of an inflammatory component plays an active role in the progression of the atherosclerotic plaque. This has led to ⁎ Corresponding author. Tel.: +972 3 6973668; fax: +972 3 6916163. E-mail address: [email protected] (J.N. Ablin). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.07.035
evolution of the concept of atherosclerosis as an extra-articular manifestation of RA (Van Doornum et al., 2002). Moreover, it has been established that effective suppression of disease activity by methotrexate can reduce mortality of rheumatoid arthritis patients (Choi et al., 2002). Endothelial progenitor cells (EPCs) are a recently identified population of circulating bone marrow-derived cells characterized by their ability to migrate into areas of increased angiogenesis and differentiate into mature endothelial cells (Asahara et al., 1997; Murohara et al., 2000). EPCs are regarded as a population with reparative capacities which have the ability to ameliorate vascular injury, thereby combating conditions of compromised tissue perfusion. The transfer of EPCs is under evaluation as a therapeutic
J.N. Ablin et al. / Life Sciences 79 (2006) 2364–2369 Table 1 Clinical characteristics of study patients Mean age (SD) Gender male/female Mean BMI (SD) Rheumatoid factor positive DAS-28 (mean, SD) Medications Hydroxychloroquine Sulfasalazine Methotrexate (average dose) Prednisone (average dose)
55.1 (11.36) 3 / 11 24.5 (3.0) 100% 5.1 (1.4) 1 / 14 (7.1%) 1 / 14 (7.1%) 10 / 14 (71.4%) (12.5 mg/week) 11 / 14 (78.6%) (6.8 mg/d)
modality potentially effective both in ischemia-related conditions such as myocardial infarction (Kawamoto et al., 2001) and in nonischemic inflammatory models such as dilated cardiomyopathy (Werner et al., 2005). EPC levels have been shown to be negatively correlated with risk factors for atherosclerosis (Hill et al., 2003), suggesting that decreased repair of vascular plaque may be the result of EPC depletion. EPCs have been identified in synovial tissue of RA patients where they may participate in angiogenesis (Ruger et al., 2004). On the other hand, it has recently been demonstrated that EPCs are depleted in the peripheral blood of RA patients (Grisar et al., 2005) and this finding has been interpreted as a possible contributing factor to the increased cardiovascular morbidity and mortality of patients suffering from this disease. Interestingly, treatment with recombinant human erythropoietin, which has been shown to improve both anemia and clinical indices of rheumatoid arthritis (Peeters et al., 1999), has recently been demonstrated to promote EPC proliferative and adhesive properties (George et al., 2005). TNFα is a central pro-inflammatory cytokine involved in pathogenesis of both RA (Choy and Panayi, 2001) and atherosclerosis (Young et al., 2002). Infliximab, a chimeric antibody targeted against TNFα, has become a mainstay of treatment in RA over the last years (Lipsky et al., 2000). Infliximab has been shown to improve endothelial function in patients with RA (Hurlimann et al., 2002; Gonzalez-Juanatey et al., 2004). Recent research implies this effect is irrespective of lipid profile (Vis et al., 2005). In the current study we have attempted to determine the effect of infliximab treatment on the number and function of EPCs in RA patients as a possible mechanism for the effect of this treatment on endothelial function. Patients and methods Study subjects Fourteen consecutive RA patients who fulfilled the American College of Rheumatology (ACR) criteria for classification of RA (Arnett et al., 1988) were recruited from the tertiary care rheumatology clinic of the Tel-Aviv Sourasky Medical Center. All participating patients had been considered eligible for infliximab treatment based on clinical assessment by the attending rheumatologist or were already being treated with infliximab. The standard treatment protocol implemented in these patients con-
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sisted of an infusion of infliximab 3 mg/kg on weeks 0, 2, 6 and subsequently every 8 weeks. Exclusion criteria included diabetes mellitus or a history of coronary artery disease, cerebrovascular disease or intermittent claudication. These patients were excluded in order to limit confounding effects on endothelial function. Although not originally defined as an inclusion criterion, all patients were sero-positive for Rheumatoid Factor (RF). This study was approved by the institutional review board and was carried out in accordance with the ethical principles of the declaration of Helsinki. All patients underwent clinical and laboratory assessment at the time of recruitment, which timed on the day of infliximab treatment and 2 weeks later. This included physical examination and a standard 28 joint count as the basis for determining disease activity 28 (DAS-28) score (Prevoo et al., 1995) and laboratory assessment of clinical activity (ESR, CRP). Subsequently, blood samples were obtained for EPC isolation before starting infliximab infusion. Isolation of EPCs and colony-forming unit assay Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density-gradient centrifugation (Sigma) from 20-ml blood samples and EPC CFU was assayed after two platings and a 9-day culture on fibronectin-coated, 24-well plates, as described (George et al., 2003; Hill et al., 2003). Colonies were manually counted in a minimum of 3 wells by two independent observers who were unaware of the patients' clinical data. Results were expressed as CFU/well in four separate wells from the same patient. Confirmation of CFU phenotype For phenotyping of endothelial characteristics of colonyforming unit (CFU), the following antibodies were used in immunofluorescence and flow cytometric analysis: rabbit polyclonal anti-Tie-2 (C-20), mouse monoclonal anti-flk-1(A-3), and goat polyclonal anti-CD31 (PECAM-1, M-20), all from Santa Cruz. Endothelial cell lineage was further confirmed by indirect immunostaining with the use of 1,1-dioctadecyl3,3,3,3-tetramethylindocarbocyanine perchlorate-acetylated low-density lipoprotein (DiI-acLDL) and co-staining with BS1 lectin (Sigma). Fibronectin adhesion assay EPCs (day 7) were washed with PBS and gently detached with 0.5 mmol/l EDTA in PBS. After centrifugation and resuspension in basal complete medium supplemented with 5% FCS, identical cell numbers were placed on fibronectin-coated culture dishes and incubated for 30 min at 37 °C (Tepper et al., 2002; George et al., 2003). Adherent cells were counted by independent blinded investigators. Results Demographic and clinical characteristics of the patients are presented in Table 1. As noted, all patients were RF positive. 10 out
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Fig. 1. A: EPC CFUs before and 14 days after treatment with infliximab. A significant increase in number of EPC CFUs, determined as described in Patients and methods, is demonstrated 14 days after treatment with infliximab. B: EPC adhesion before and 14 days after treatment with infliximab. A significant increase is observed in EPC adhesion as measured by adherence to fibronectin (see Patients and methods), 14 days after treatment with infliximab. C: Effect of infliximab treatment on EPC differentiation. EPC differentiation was measured by two separate observers and scored on a semi-quantitative scale. A significant increase in EPC differentiation is observed after treatment with infliximab. D: Reduction of DAS-28 after infliximab treatment. Clinical assessment of patients was documented by determination of the DAS-28 score. The figure demonstrates the clinical response to infliximab reflected by a significant reduction of the DAS-28 14 days after treatment with infliximab.
J.N. Ablin et al. / Life Sciences 79 (2006) 2364–2369 Table 2 EPC colonies before, and 2 weeks after treatment with infliximab Patient number
Colonies before treatment
Colonies after treatment
Δ (absolute)
Δ (%)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
25 31 97 24 45 47 30 69 57 32 18 99 20 58
32 59 102 52 42 75 64 75 52 48 32 120 55 62
7 28 5 28 −3 28 34 6 −5 16 14 21 35 4
28 90.3 5.6 117 − 6.7 59.6 113 8.7 − 8.8 50 77.8 21.2 175 6.9
of 14 patients (71.4%) were treated with methotrexate while 11 / 14 (78.6%) were on corticosteroids. The average duration of prior treatment with infliximab was 9.6 months (range — 1–32 months, SD — 26.2). A significant 33.4% increase [from 46.6(SD — 26.7) to 62.1(SD — 24.7) CFU/well; pb 0.001] in EPC levels was observed 14 days after infliximab treatment (Fig. 1A). Table 2 presents the absolute and percentage change in EPC CFUs following treatment with infliximab. Aiming to determine whether infliximab treatment influences the functional properties of EPC, we performed adhesion assays. A 37.6% increase was observed in mean number of adhering EPC from 75.43 (SD — 41.0) to 103.79 (SD — 42.5) cells per plate ( pb 0.001) (Fig. 1B). A 60% increase was noted in the EPC differentiation scale, from 2.5 (SD — 1.6) to 4.0 (SD — 1.1) ( pb 0.002) (Fig. 1C). These changes were associated with a 17.5% decrease in the DAS-28 from 5.1 (SD — 1.4) to 4.2 (SD — 1.1) ( p b 0.0001) (Fig. 1D), reflecting the clinical response to infliximab. A significant correlation was observed between the change in DAS-28 and the change in EPC CFU (Fig. 2) implying an interrelation between this clinical index and the physiologic effect of anti-TNFα treatment on EPCs.
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Herbrig et al. have shown a clear correlation between endothelial dysfunction in RA patients and impaired function of EPCs (Herbrig et al., 2005). Depletion of EPCs in RA has previously been shown by Grisar et al. who studied 52 patients at different levels of disease activity and treated with a variety of DMARDs. They observed an inverse correlation between the number of EPCs and disease activity. EPC levels were not affected by the use of DMARDs except for anti-TNFα therapy, which, in 4 patients, was associated with a higher number of EPCs, similar to healthy controls (Grisar et al., 2005). Our study confirms and further substantiates this observation, demonstrating the dynamic effect of infliximab on both the number and function of EPCs, in consistence with the favorable effect of infliximab on endothelial function of RA patients (Hurlimann et al., 2002; Gonzalez-Juanatey et al., 2004). Our results demonstrate the capacity of anti-TNFα treatment to improve EPC levels and function in parallel with their clinical anti-inflammatory effect. Since EPCs are considered reparative cells, capable of ameliorating ischemic damage (Takahashi et al., 1999), this finding implies a potential mechanism through which this potent anti-inflammatory treatment may act to decrease the vasculopathy involved in RA (and possibly other chronic inflammatory conditions) and which may translate into reduced cardiovascular morbidity and mortality. Our results offer a novel viewpoint on the effect of anti-inflammatory treatment on vascular biology in rheumatoid arthritis. Although the link between this prototypical inflammatory joint disease and both atherosclerosis and cardiovascular morbidity and mortality has been epidemiologically well established, the role of endothelial progenitor cells, and the effect of anti-inflammatory treatment on this population have not been previously studied. Hence, the improved EPC levels and function observed here could provide a plausible mechanistic explanation. Moreover, these results could point towards the potential to develop novel therapeutic interventions in rheumatoid arthritis and in similar inflammatory disorders which would specifically target mediators which down-regulate EPCs. This possibility is highlighted by the recent results demonstrating that TNFα can directly inhibit EPCs (Seeger et al., 2005). An alternative approach would be to pursue medications which, similar to HMG-CoA-reductase inhibitors
Discussion In this study, we have shown that a single dose of infliximab treatment improved the number and functional properties of EPCs in patients with RA, in parallel with the expected clinical improvement. A significant correlation was observed between the extent of clinical improvement (reflected by the DAS-28) and the level of increase in the number and function of EPC CFUs. This clear effect suggests a potential novel mechanism which may explain the salutory effect of anti-inflammatory treatment on the cardiovascular risk associated with rheumatoid arthritis. Rheumatoid arthritis is characterized by impaired endothelial function (Vaudo et al., 2004; Herbrig et al., 2005). Young to middle aged patients with RAwith low disease activity, free from cardiovascular risk factors and cardiovascular disease, have altered endothelial reactivity, probably related to the underlying inflammatory condition (Vaudo et al., 2004). Furthermore,
Fig 2. Correlation between improvement in DAS-28 and change in EPC CFUs. Patients with moderate to severe RA were evaluated for their DAS-28 scores and EPC numbers prior to and after a course of infliximab. A correlation was sought between EPC numbers and DAS-28 as described in Patients and methods.
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(Assmus et al., 2003) and erythropoietin (George et al., 2005), directly stimulate EPCs. Notably, EPCs play additional roles in rheumatoid arthritis. Ruger et al have demonstrated the presence of endothelial progenitor cell in synovial tissue of RA patients (as well as osteoarthritis) suggesting a role for these cells in the neovascoulogenesis involved in the formation of panus (Ruger et al., 2004). Thus, while EPCs are presumed to be systemically protective against ischemia, they may simultaneously facilitate the proliferative process responsible for cartilage destruction in RA. Moreover, TNFα may play a complex role in this cascade. Thus, while TNFα has a pro-apoptotic effect on bone marrow-derived progenitor cells (thus contributing to inflammatory induced anemia) it appears to protect inflammatory cells within the RA joint (e.g. macrophages and synovial fibroblasts), thus contributing to joint destruction in RA (Liu and Pope, 2003). TNFα also appears to down-regulate EPCs through additional pathways not dependent on apoptosis (Seeger et al., 2005). Since the measured serum levels of EPCs may represent the product of a balance between bone marrow production and mobilization and peripheral apoptosis and breakdown, a logical subsequent step after the current study would be to try and elucidate the correlation between levels of bioavailable TNFα and the effect on EPCs. Determining such levels would however be particularly difficult due to the binding of TNFα by infliximab, a specific monoclonal antibody aimed against that cytokine. In our study, we employed the CFU method for assessment of EPC numbers. This method employs evaluation of formation of clusters of colonies consisting of a core of concentrated cells with eccentrically ‘protruding’ spindle shaped cells. These colonies are routinely stained for several endothelial cell markers such as Tie-1, KDR and CD31 and the results are also crossvalidated by double staining for a lectin (BS-1) and a receptor for acetylated LDL. We are aware of the current debate as to the accurate methodology with regard to assessment of EPC num bers. However, neither consensus nor standardization has hitherto been achieved and several groups are employing the CFU method (Hill et al., 2003; George et al., 2003; Assmus et al., 2003), whereas others favor analysis of FACS markers that are equally problematic (some regard EPC as CD34+CD133+, others CD34+/KDR and yet others prefer the CD133/KDR). In a very recent study (George et al., 2006), we have compared all these methods and found that they did not correlate, yet CFUs were correlated with functional properties consistent with proliferation of endothelial like cells. In conclusion, we have found that infliximab enhances the number and function of EPCs in patients with moderate to severe rheumatoid arthritis in correlation with the clinical improvement. This finding may be suggestive of a novel mechanism by which anti-inflammatory treatment in general and anti-TNFα treatment in particular may alter the increased cardiovascular morbidity and mortality associated with rheumatoid arthritis. References Arnett, F.C., Edworthy, S.M., Bloch, D.A., McShane, D.J., Fries, J.F., Cooper, N.S., Healey, L.A., Kaplan, S.R., Liang, M.H., Luthra, H.S., et al., 1988. The American
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