Increased Levels of Circulating Endothelial-Derived Microparticles and Small-Size Platelet-Derived Microparticles in Psoriasis
2011; Elsevier BV; Volume: 131; Issue: 7 Linguagem: Inglês
10.1038/jid.2011.57
ISSN1523-1747
AutoresF. Pelletier, Francine Garnache‐Ottou, Fanny Angelot, Sabeha Biichlé, Chrystelle Vidal, Philippe Humbert, Philippe Saas, Estelle Seillès, F. Aubin,
Tópico(s)Psoriasis: Treatment and Pathogenesis
ResumoAnnexin-V Dermatology Life Quality Index endothelial-derived microparticle microparticle Psoriasis Area Severity Index platelet-derived microparticle tumor necrosis factor-alpha TO THE EDITOR Microparticles (MPs) are membrane vesicles of 0.1 to 1μm diameter generated by budding or shedding from the plasma membrane and released by any cell type into the vascular compartment during activation or apoptosis (Théry et al., 2009Théry C. Ostrowski M. Segura E. Membrane vesicles as conveyors of immune responses.Nat Rev Immunol. 2009; 9: 581-593Crossref PubMed Scopus (2577) Google Scholar). Membrane lineage markers of their originating cell permit to distinguish several circulating MPs, such as platelet-derived MPs (PMPs) or endothelial cell-derived MPs (EMPs). Microparticles are involved in inflammatory and auto-immune diseases, as well as in cardiovascular disorders and the metabolic syndrome (Helal et al., 2010Helal O. Defoort C. Robert S. et al.Increased levels of microparticles originating from endothelial cells, platelets and erythrocytes in subjects with metabolic syndrome: relationship with oxidative stress.Nutr Metab Cardiovasc Dis. 2010Google Scholar; Leroyer et al., 2010Leroyer A.S. Anfosso F. Lacroix R. et al.Endothelial-derived microparticles: biological conveyors at the crossroad of inflammation, thrombosis and angiogenesis.Thromb Haemost. 2010; 104: 456-463Crossref PubMed Scopus (139) Google Scholar). MP implication in psoriasis pathophysiology is suggested by several features: (i) the pathogenic role of TNF-α (Nestle et al., 2009Nestle F.O. Kaplan D.H. Barker J. Psoriasis.N Engl J Med. 2009; 361: 496-509Crossref PubMed Scopus (1942) Google Scholar), a powerful in vitro inducer of EMP generation (Combes et al., 1999Combes V. Simon A.C. Grau G.E. et al.In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant.J Clin Invest. 1999; 104: 93-102Crossref PubMed Scopus (632) Google Scholar); (ii) the presence of activated endothelial cells within cutaneous lesions (Nestle et al., 2009Nestle F.O. Kaplan D.H. Barker J. Psoriasis.N Engl J Med. 2009; 361: 496-509Crossref PubMed Scopus (1942) Google Scholar) and EMP generation that may reflect endothelium aggression; and (iii) the presence of platelet activation in psoriasis (Garbaraviciene et al., 2010Garbaraviciene J. Diehl S. Varwig D. et al.Platelet P-selectin reflects a state of cutaneous inflammation: possible application to monitor treatment efficacy in psoriasis.Exp Dermatol. 2010; 19: 736-741Crossref PubMed Scopus (42) Google Scholar; Tamagawa-Mineoka et al., 2010Tamagawa-Mineoka R. Katoh N. Kishimoto S. Platelet activation in patients with psoriasis: increased plasma levels of platelet-derived microparticles and soluble P-selectin.J Am Acad Dermatol. 2010; 62: 621-626Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) that may generate PMPs (Italiano et al., 2010Italiano Jr., J.E. Mairuhu A.T. Flaumenhaft R. Clinical relevance of microparticles from platelets and megakaryocytes.Curr Opin Hematol. 2010; 36: 881-887Google Scholar). Moreover, the association between psoriasis and atherosclerotic risk factors (Boehncke et al., 2010Boehncke W.H. Boehncke S. Schön M.P. Managing comorbid disease in patients with psoriasis.BMJ. 2010; 340: b5666Crossref PubMed Scopus (103) Google Scholar) may be related to excessive MP production. Based on this, we investigated circulating MPs in psoriatic patients. The study design was approved by the local research ethics committee and written informed consent was provided before enrolment. The study adhered to the Declaration of Helsinki Principles. Fifty-two psoriatic patients analyzed before the introduction of systemic treatments and 30 healthy blood donors were investigated (Table 1). Patients with other dermatosis, with a history of cardiovascular disease, diabetes, chronic renal failure, or chronic inflammatory disease, were excluded. The severity of psoriasis was evaluated by the Psoriasis Area Severity Index (PASI) and the Dermatology Life Quality Index (DLQI) scores. We adapted the MP quantification method developed by Robert et al., 2009Robert S. Poncelet P. Lacroix R. et al.Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC 500 routine flow cytometer: a first step towards multicenter studies?.J Thromb Haemost. 2009; 7: 190-197Crossref PubMed Scopus (249) Google Scholar. Plasma was separated from whole blood by centrifugation at 1500g for 15minutes. Recovered plasma was centrifuged for 2min at 13,000g to remove residual cells and platelets. Microparticles were labeled using FITC-conjugated Annexin-V (AV) (BD Biosciences, Le Pont de Claix, France) and fluorescent mAbs were added to identify the MP cellular origin. The following mAbs were used: phycoerythrin (PE)-conjugated CD31 and PE-Texas Red-x (ECD)-conjugated CD41 (Beckman Coulter, Villepinte, France), PE-conjugated CD62E (BD Biosciences), and fluorescent-conjugated isotype control mAbs. After 30-minute incubation and addition of Flow-Set™ fluorosphere beads (Beckmann Coulter), samples were analyzed using a NAVIOS cytometer (Beckman Coulter). MP number was calculated on the basis of the known number of Flow-Set™ beads added to the sample. EMPs were identified as CD31+/CD41−/AV+/− or CD62E+/CD41−/AV+/− events, and PMPs as CD31+/CD41+/AV+/− events (Figure 1a–e, Supplementary Table S1 online). Two MP-size regions were determined and standardized daily with Megamix fluorescent beads (Biocytex, Marseille, France) containing two types of beads with a defined size (0.5 and 0.9μm diameter, respectively). Two nonparametric statistical tests (Wilcoxon and Kruskal–Wallis tests) were used to compare the means of circulating MP counts.Table 1Main clinical characteristics of subjects and plasma levels of EMPs, PMPs, and AV+ MPs in patients with psoriasis and in healthy control subjectsControl, n=30Psoriasis, n=52PAge (years)aResults are expressed as median (range).50 (18–65)51.8 (25–96)NSSex (male/female)15/1529/23NSC-reactive protein (mgl−1)aResults are expressed as median (range).1.0 (1–1)5.46 (1–11)NDPASI scoreaResults are expressed as median (range).ND18 (14–25)NDDLQIaResults are expressed as median (range).ND12.5 (0–26)NDEMPsbResults are expressed as the mean number±standard deviation of microparticles per plasma μl (range). Microparticle size (that is, between 0.5 and 0.9μm or <0.5μm) is also considered. Bold values are statistically significant. Total CD31+/CD41− EMPs12±10 (2–44)bResults are expressed as the mean number±standard deviation of microparticles per plasma μl (range). Microparticle size (that is, between 0.5 and 0.9μm or <0.5μm) is also considered. Bold values are statistically significant.80±121 (3–543)0.0002 0.5–0.9μm CD31+/CD41+ EMPs3±4 (0–15)26±52 (0–222)0.0035 <0.5μm CD31+/CD41− EMPs8±7 (0–29)54±82 (0–433)<0.0001 Total CD62E+/CD41− EMPs3±2 (0–12)5±5 (0–24)0.09 0.5–0.9μm CD62E+/CD41− EMPs1±1 (0–6)2±3 (0–16)0.21 <0.5μm CD62E+/CD41 EMPs2±2 (0–5)4±4 (0–13)0.006PMPsbResults are expressed as the mean number±standard deviation of microparticles per plasma μl (range). Microparticle size (that is, between 0.5 and 0.9μm or <0.5μm) is also considered. Bold values are statistically significant.(CD31+/CD41+)4,515±3,787 (829–16279)bResults are expressed as the mean number±standard deviation of microparticles per plasma μl (range). Microparticle size (that is, between 0.5 and 0.9μm or <0.5μm) is also considered. Bold values are statistically significant.5,621±7,030 (340–31,054)0.35 0.5–0.9μm PMPs3,006±2,921 (230–11332)2,092±2,331 (86–10,049)0.12 <0.5μm PMPs1,508±964 (321–4947)3,528±5,122 (254–22,291)0.007AV+ MPsbResults are expressed as the mean number±standard deviation of microparticles per plasma μl (range). Microparticle size (that is, between 0.5 and 0.9μm or <0.5μm) is also considered. Bold values are statistically significant.3,052±2,850 (422–10568)bResults are expressed as the mean number±standard deviation of microparticles per plasma μl (range). Microparticle size (that is, between 0.5 and 0.9μm or <0.5μm) is also considered. Bold values are statistically significant.3,177±2,568 (129–11,537)0.83 0.5–0.9μm AV+ MPs2,020±1,807 (153–6435)1,428±1,391 (43–7,280)0.10 <0.5μm AV+ MPs1,157±860 (240–4144)1,623±1,631 (87–6,667)0.09Abbreviations: AV, Annexin-V; DLQI, Dermatology Life Quality Index; EMPs, endothelial-derived microparticles; MPs, microparticles; ND, not determined; NS, nonsignificant; PASI, Psoriasis Area Severity Index; PMPs, platelet-derived microparticles.a Results are expressed as median (range).b Results are expressed as the mean number±standard deviation of microparticles per plasma μl (range). Microparticle size (that is, between 0.5 and 0.9μm or <0.5μm) is also considered.Bold values are statistically significant. Open table in a new tab Download .pdf (.01 MB) Help with pdf files Supplementary Information Abbreviations: AV, Annexin-V; DLQI, Dermatology Life Quality Index; EMPs, endothelial-derived microparticles; MPs, microparticles; ND, not determined; NS, nonsignificant; PASI, Psoriasis Area Severity Index; PMPs, platelet-derived microparticles. Circulating EMP levels were increased in psoriatic patients (Table 1, Figure 1f and g). Circulating CD31+41− EMPs were statistically higher compared with EMP levels in the control group (80μl−1, ±121 vs. 12μl−1, ±10; P=0.0002). A trend toward significance was observed for CD62E+ EMPs (5μl−1, ±5 in patients vs. 3μl−1, ±2 in healthy subjects; P=0.09). When focusing on the smaller-size EMPs, we found a significantly higher number of both CD31+/CD41− EMPs (54μl−1, ±82 vs. 8μl−1, ±7; P<0.0001) and CD62E+ EMPs (4μl−1, ±4 vs. 2μl−1, ±2; P=0.006) in psoriatic patients. When considering larger-size EMPs (≥0.5μm), we detected a significantly higher number of CD31+/CD41− EMPs (30μl−1, ±52) in patients than in the control group (3μl−1, ±4; P=0.0035). No significant difference was observed for CD62E+ EMPs. No correlation with psoriasis severity (assessed by PASI and DLQI scores) was observed. No difference was found in circulating CD31+/CD41+ PMP levels between psoriatic patients (5,621μl−1, ±7,030) and healthy controls (4,515μl−1, ±3,787; P=0.35). However, smaller-size PMP levels were significantly higher in psoriatic patients than in healthy patients (3,528μl−1, ±5,122 vs. 1,508μl−1, ±964, P=0.007). We did not observe any correlation with psoriasis severity. Total circulating AV+MP counts (whatever their origin) in psoriatic patients (3,052μl−1, ±2,852) did not significantly differ from those quantified in the control group (3,177μl−1, ±2,568, P=0.83). There was no difference depending on the size of AV+MPs (Table 1). Our study demonstrates that circulating EMPs and small-size PMPs are significantly increased in psoriatic patients as compared with healthy subjects. Tamagawa-Mineoka et al., 2010Tamagawa-Mineoka R. Katoh N. Kishimoto S. Platelet activation in patients with psoriasis: increased plasma levels of platelet-derived microparticles and soluble P-selectin.J Am Acad Dermatol. 2010; 62: 621-626Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar previously showed that plasma PMP levels were significantly increased in 21 psoriasis patients. The discrepancy with our results may be related to the methods used for PMP quantification: ELISA versus flow cytometry. Cytometry permits to determine MP size (Orozco and Lewis, 2010Orozco A.F. Lewis D.E. Flow cytometric analysis of circulating microparticles in plasma.Cytometry A. 2010; 77: 502-514Crossref PubMed Scopus (243) Google Scholar). Small-size PMPs –identified here– may reflect platelet activation. Several evidences suggest that the majority of PMPs circulating in healthy subjects are derived from megakaryocytes, while PMPs derived from activated platelets have been separated into four size classes, including small-size PMPs (Italiano et al., 2010Italiano Jr., J.E. Mairuhu A.T. Flaumenhaft R. Clinical relevance of microparticles from platelets and megakaryocytes.Curr Opin Hematol. 2010; 36: 881-887Google Scholar), with different functional effects on platelets and endothelial cells (Dean et al., 2009Dean W.L. Lee M.J. Cummins T.D. et al.Proteomic and functional characterisation of platelet microparticle size classes.Thromb Haemost. 2009; 102: 711-718PubMed Google Scholar). Soluble P-selectin level, a platelet-activation hallmark, is a biomarker for inflammation in psoriasis (Garbaraviciene et al., 2010Garbaraviciene J. Diehl S. Varwig D. et al.Platelet P-selectin reflects a state of cutaneous inflammation: possible application to monitor treatment efficacy in psoriasis.Exp Dermatol. 2010; 19: 736-741Crossref PubMed Scopus (42) Google Scholar). Increased EMP levels could be explained by endothelial cell activation by TNF-α, since TNF-α favors in vitro EMP generation (Combes et al., 1999Combes V. Simon A.C. Grau G.E. et al.In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant.J Clin Invest. 1999; 104: 93-102Crossref PubMed Scopus (632) Google Scholar). We showed that EMPs induce plasmacytoid dendritic cell (pDC) maturation (Angelot et al., 2009Angelot F. Seillès E. Biichle S. et al.Endothelial cell-derived microparticles (EMP) induce plasmocytoid dendritic cell maturation: potential implications in inflammatory diseases.Haematologica. 2009; 94: 1502-1512Crossref PubMed Scopus (73) Google Scholar). As pDCs are essential to drive psoriasis development (Nestle et al., 2005Nestle F.O. Conrad C. Tun-Kyi A. et al.Plamacytoid predendritic cells initiate psoriasis through interferon-α production.J Exp Med. 2005; 202: 135-143Crossref PubMed Scopus (798) Google Scholar), EMPs could represent an activating factor for pDCs and thus contribute to inflammation. Systemic inflammation in turn causes insulin resistance in psoriasis, a state in which insulin is proatherogenic (Boehncke et al., 2007Boehncke S. Thaci D. Beschmann H. et al.Psoriasis patients show signs of insulin resistance.Br J Dermatol. 2007; 157: 1249-1251Crossref PubMed Scopus (204) Google Scholar). As both small-size PMPs (Dean et al., 2009Dean W.L. Lee M.J. Cummins T.D. et al.Proteomic and functional characterisation of platelet microparticle size classes.Thromb Haemost. 2009; 102: 711-718PubMed Google Scholar) and EMPs (Leroyer et al., 2010Leroyer A.S. Anfosso F. Lacroix R. et al.Endothelial-derived microparticles: biological conveyors at the crossroad of inflammation, thrombosis and angiogenesis.Thromb Haemost. 2010; 104: 456-463Crossref PubMed Scopus (139) Google Scholar) express procoagulant phosphatidylserine and tissue factor activities, increased small-size PMP and EMP levels may contribute to accelerated atherosclerosis in psoriatic patients. However, the clinical relevance of our results remains still disputed: does EMP generation represent an epiphenomenon related to endothelium activation by TNF-α or do EMPs have a role in psoriasis pathophysiology –through pDC activation– leading to accelerated atherosclerosis? The authors would like to thank Antoine Pachéco (Beckman Coulter), Elisabeth Homassel, and Stéphane Robert for technical support and manuscript editing. They are also grateful to Françoise Dignat-George for helpful discussion. This work was supported by the Fondation Transplantation (grants ET-051222, ET-060934, and ET-070636), the “Société de Recherche Dermatologique (2008)”, the University of Franche-Comté (2008), and the University Hospital of Besançon (APICHU 2008). Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
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