Osteopontin (OPN) is a multifunctional glycophosphoprotein secreted by many cell types, including osteoblasts, lymphocites, macrophages, epithelial cells, and vascular smooth muscle cells. It has been implicated in many physiological and pathological processes, such as cell-mediated immunity, inflammation, cell survival, and tumor invasion and metastasis. Osteopontin has multiple emerging roles in cutaneous biology and pathology and OPN involvement has been emphasized in Th1-mediated diseases such as psoriasis. Alopecia areata (AA) is a form of non-scarring hair loss affecting anagen stage hair follicles with a multifactorial autoimmune pathogenesis characterized by a prevalent Th1 cytokine profile. Given the role of osteopontin in Th1-mediated inflammation, we have postulated that OPN may be involved in AA pathogenesis. The aim of our study was to investigate plasma OPN level in alopecia areata before and after DPCP treatment. Our results showed that OPN plasma levels in patients with alopecia areata were higher than in healthy controls, but patients achieving complete recovery after DPCP treatment did not show a statistically significant reduction of OPN plasma levels.</p

Anna Campanati

Annamaria Offidani

Giulia Ganzetti

Katia Giuliodori

Marina Brugia

Massimo Tocchini

Oriana Simonetti

Vitangelo Scocco

English

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19ACTA DERMATOVENEROLOGICA CROATICAOsteopontin: A New Facilitating Factor in Alopecia Areata Pathogenesis?Giulia Ganzetti1, Oriana Simonetti1, Anna Campanati1, Katia Giuliodori1, Vitangelo Scocco2, Marina Brugia2, Massimo Tocchini2, Anamaria Offidani11Dermatological Clinic, Department of Clinical and Molecular Medicine, Polytechnic Marche University, Ancona, Italy; 2 Laboratory Analysis, Ospedali Riuniti “Umberto I”, Ancona, ItalyCorresponding author: Anna Campanati, MDVia conca 7160020 AnconaItalyannina.campanati@gmail.com Received: August 30, 2013Accepted: July 31, 2014Acta Dermatovenerol Croat 2015;23(1):19-22      SHORT SCIENTIFIC COMMUNICATIONAbstrACt Osteopontin (OPN) is a multifunctional glycophosphoprotein secreted by many cell types, including osteoblasts, lymphocites, macrophages, epithelial cells, and vascular smooth muscle cells. It has been implicated in many physiological and pathological processes, such as cell-mediated immunity, inflammation, cell surviv-al, and tumor invasion and metastasis. Osteopontin has multiple emerging roles in cutaneous biology and pathology and OPN involvement has been emphasized in Th1-mediated diseases such as psoriasis. Alopecia areata (AA) is a form of non-scar-ring hair loss affecting anagen stage hair follicles with a multifactorial autoimmune pathogenesis characterized by a prevalent Th1 cytokine profile. Given the role of osteopontin in Th1-mediated inflammation, we have postulated that OPN may be involved in AA pathogenesis. The aim of our study was to investigate plasma OPN level in alopecia areata before and after DPCP treatment. Our results showed that OPN plasma levels in patients with alopecia areata were higher than in healthy con-trols, but patients achieving complete recovery after DPCP treatment did not show a statistically significant reduction of OPN plasma levels.Key wOrds: ostepontin, alopecia areata, diphenylciclopropenoneINtrOduCtIONAlopecia areata (AA) is a type of non-scarring hair loss affecting anagen stage hair follicles with a multi-factorial autoimmune pathogenesis and an unknown etiology (1).It affects about 2-3% of new patients attending Dermatology Clinics, presenting a wide range of clini-cal heterogeneity (1).AA involves the scalp and any hair-bearing body surface, with possible nail localization in 10% of pa-tients. A wide spectrum of clinical presentation can oc-cur – from single or multiple patches of hair loss to a complete hair loss on the scalp (alopecia totalis) or the entire body (alopecia universalis); the affected skin ap-pears normal or slightly erythematous and edematous. Most cases of alopecia areata resolve spontaneously within 1 year, but up to 25% develop into severe forms such as alopecia totalis and alopecia universalis (1,2).Topical immunotherapy with 2,3-diphenilcyclo-propenone (DPCP) has shown to be one of the most effective treatments for severe and refractory AA and to be able to modify the “cytokine storm” (3). Although many molecular pathways are impli-cated in AA immune-pathogenesis, Th1 cytokine pro-file seems to play a crucial role: a greater expression of interferon gamma (IFN-γ), interleukin 1 (IL-1), and tumor necrosis factor alpha (TNF-α) has been exten-sively shown in serum and tissue of patients with AA compared to healthy controls (1-4).20 ACTA DERMATOVENEROLOGICA CROATICAOsteopontin (OPN), also known as early T lym-phocyte activation 1 (Eta-1), is a phosphorylated acid glycoprotein firstly identified as a bone component involved in bone formation and calcification. It is se-creted by osteoblasts, lymphocytes, macrophages, keratinocytes, and vascular smooth cells (4,5). Osteo-pontin is also implicated in cell-mediated immunity, cell survival, inflammation, and tumor invasion and metastases (6-7).High circulating OPN levels have been reported in several dermatological and non-dermatological dis-eases, but data on OPN expression in patients with alopecia areata are not available in the literature (8).Therefore, given the role of osteopontin in Th1-mediated inflammation, we have postulated that OPN may be involved in the pathogenesis of AA.The aim of our study was to investigate plasma OPN level in alopecia areata before and after DPCP treatment.PAtIeNts ANd MethOdsdesign of the studyThe study is a retrospective case-control analysis approved by the Polytechnic Marche University Ethi-cal Committee and conducted in accordance with the Declaration of Helsinski (9).PopulationAmong blood samples stored at -80°C in our se-rum/plasma bank, we selected the following samples: 40 patients affected by alopecia areata (23 men and 17 women; mean age 40.25±6.35; range 20-55), both before (T0) and after twelve months (T12) of treat-ment with DPCP, and twenty controls obtained from healthy subjects matched for sex and age.Blood samples of patients who had had a clini-cal recurrence of disease after DPCP discontinuation were also retrieved.Although not an exclusion criterium, neither fa-miliar history of AA nor personal history of atopy or autoimmune disease, including thyroiditis, had been reported in the patient group.diagnosis of alopecia areataDiagnosis of alopecia areata had been made by a trained dermatologist based on the clinical evalu-ation of a diffuse or patchy alopecia and the presence of exclamation mark hairs. In patchy AA the severity of hair shedding had been evaluated by a pull test on three separate areas of the scalp: an extraction of six or more hairs was considered a positive pull test (10).The images stored from a previous video-derma-toscopic evaluation were retrieved to evaluate the most common signs of alopecia areata: yellow dots (hair follicles filled with hyperkeratotic plugs), black dots (hair follicles containing cadaverized hairs), bro-ken hairs, exclamation point hairs, clustered short vellus hairs, and their changes after treatment with DPCP (11).All the enrolled patients with AA had not respond-ed to any previous topical and intralesional cortico-steroid therapy; thus after a four-weeks wash out period, they received DPCP following the protocol proposed by Happle et al. (12).The DPCP concentrations had been progressively increased until erythema and/or pruritus appeared and was maintained for 36-48 hours after application. This dose was then considered the gold standard, and patients were asked to apply it until complete recovery.Figure 1. Osteopontin plasma levels in patients with AA in comparison with healthy controls.Figure 2. Osteopontin plasma levels in patients with AA before and after DPCP treatment in comparison with healthy controls.Ganzetti et al. Acta Dermatovenerol CroatOsteopontin in alopecia areata    2015;23(1):19-2221ACTA DERMATOVENEROLOGICA CROATICALaboratory assaysVenous blood samples had been drawn after an overnight fast at baseline (T0, before DPCP treatment) and after twelve months of DPCP application. OPN quantitative determination was done through a solid phase sandwich ELISA kit on EDTA plasma samples. This assay kit precoats a monoclonal antibody specific for OPN onto a microplate to bind OPN in the samples. All blood samples had been pre-served at -80°C. Plasma samples were dilated and the immunoassay was performed according to the man-ufacturer’s instructions. statistical analysisAll data were analyzed using Graph-Pad Prism (version 5.0, El Camino REAL, San Diego, CA) and QuickCalcs software package. Data were expressed as means±SD. The normal distribution of continuous variables was verified with the Kolmogorov-Smirnov test, and statistical analyses included the Mann-Whit-ney test for continuous variables. Linear regression was used to model the relationship between vari-ables. For all the analyses, a P-value <0.05 was consid-ered to be statistically significant.resuLtsIn the group of patients with AA, thirty-one pa-tients had patchy alopecia (<50% scalp involvement) and nine alopecia totalis; the mean time of the onset of the disease was 4.05±2.37 months. In three patients treatment was discontinued for severe acute contact dermatitis after sensitization with DPCP 2%. Thirty-seven patients completed the treatment, showing initial hair regrowth after twelve weeks of DPCP and obtaining a complete recovery af-ter a mean period of 12 months. Thirty-three patients maintained these satisfac-tory results for almost twelve months following the discontinuation of DPCP treatment; four patients with patchy AA developed a recurrence three months after DPCP discontinuation.At baseline, the mean plasma OPN value in pa-tients with AA was significantly higher than in healthy controls (85.25±46.48 ng/mL vs. 46.60±26.33 ng/mL; P=0.0077) (Figure 1); however, plasma levels of OPN did not correlate with severity and duration of AA.There was no statistically significant decrease of mean plasma levels of OPN (73.59±27.48 vs 85.25±46.48, P>0.05) after DPCP treatment, although it was significantly higher than in healthy subjects (73.59±27.48 vs. 46.60±26.33 ng/mL, P=0.0084) (Figure 2).Additionally, in the subgroup of patients with recurrent AA, relapse was associated with increased plasma levels of OPN, levels of which tended towards those found at T0 (mean: 76.00±13.67 ng/mL).dIsCussIONOsteopontin is a multifunctional protein that plays an important role in inflammatory processes inhibit-ing Th2 response and promoting Th1 cytokine func-tion, with a crucial role in leukocyte migration (7,8).Our study shows that OPN plasma levels in pa-tients with AA were higher than in healthy controls, and it could be hypothesized that osteopontin has a role in the pathogenesis of alopecia areata, amplify-ing the inflammatory cascade.Alopecia areata is considered a Th1-mediated skin disease, for which topical immunotherapy with 2,3-diphenilcyclopropenone (DPCP) has shown to be one of the most effective treatments, even though thera-peutic response differs from patient to patient. DPCP is a potent contact sensitizer, whose mechanism of action in AA is not yet completely understood; how-ever, a hypothesis based on cytokine inhibitors pro-poses a possible interference of DPCP with pre-exist-ing pro-inflammatory cytokines and their continued production by follicular keratinocytes (5,10).In our case series, patients obtaining complete recovery after DPCP treatment did not show a sta-tistically significant reduction of OPN plasma levels. It could be inferred that DPCP acts primarily on the skin without any significant interference on systemic levels.  However, it is interesting to note that OPN plasma level increases in patients with alopecia areata recur-rence after DPCP discontinuation, whereas a statisti-cal significant difference between OPN values has not been found before and after the DPCP treatment. Based on these data, it is not possible assuming OPN as a predictive marker of disease, taking into account the unpredictability and intensity of the relapse of AA (13).Furthermore, OPN expression has been shown to be linked to vitamin D levels in bone and kidney-stone formation. Currently the primary deficit of vi-tamin D is considered crucial for the development of autoimmune disease, such as alopecia areata. Thus, we could hypothesize that increased serum levels of OPN could be a primitive facilitating factor in AA pathogenesis, possibly acting together with vitamin D deficiency (14,15).Ganzetti et al. Acta Dermatovenerol CroatOsteopontin in alopecia areata    2015;23(1):19-2222 ACTA DERMATOVENEROLOGICA CROATICACONCLusIONThis is the first pilot study suggesting a possible involvement of osteopontin in AA pathogenesis; fur-ther and larger prospective studies are needed in or-der to achieve in-depth knowledge of the role of OPN in AA.references1. Gregoriou S, Papafragkaki D, Kontochristopoulos G, Rallis E, Kalogeromitros D, Rigopoulos D. Cy-tokines and other mediators in alopecia areata. Mediators Inflamm 2010;2010:928030.2. Ganzetti G, Campanati A, Offidani A. Alopecia Areata: a possible extraintestinal manifestation of Crohn’s disease. J Crohns Colitis 2012;6:962-3.3.  Ferretti G, Bacchetti T, Campanati A, Simonetti O, Liberati G, Offidani A. Correlation between lipoprotein(a) and lipid peroxidation in psoriasis: role of the enzyme paraoxonase-1. Br J Dermatol. 2012;166:204-74.  Campanati A, Giuliodori K, Ganzetti G, Liberati G, Offidani A. A patient with psoriasis and viti-ligo treated with etanercept. Am J Clin Dermatol. 2010;11 suppl 1:46-8. 5. Alkhalifah A, Alsantali A, Wang E, McElwee KJ, Shapiro J. Alopecia areata update: part II. Treat-ment. J Am Acad Dermatol 2010;62:191-202.6. Denhardt DT, Noda M, O’Regan AW, Pavlin D, Ber-man JS. Osteopontin as a means to cope with en-vironmental insults: regulation of inflammation, tissue remodeling, and cell survival. J Clin Invest 2001;107:1055-61. 7. Ashkar S, Weber GF, Panoutsakopoulou V, Sanchi-rico ME, Jansson M, Zawaideh S, et al. Eta-1 (osteo-pontin): an early component of type-1 (cell-medi-ated) immunity. Science 2000;4:860-4.8. Buback F, Renkl AC, Schulz G, Weiss JM. Osteo-pontin and the skin: multiple emerging roles in cutaneous biology and pathology. Exp Dermatol 2009;18:750-9.9. World Medical Association Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Patients. Adopted by the 18th WMA Ge-neral Assembly 1964 and amended by the 29th, 35th, 41st and 48th WMA General Assemblies.10. Gordon KA, Tosti A. Alopecia: evaluation and treat-ment. Clin Cosmet Investig Dermatol 2011;4:101-6.11. Ganzetti G, Campanati A, Simonetti O, Cataldi I, Giuliodori K, Offidani AM. Videocapillaroscopic pattern of alopecia areata before and after diphe-nylciclopropenone treatment. Int J Immunopat-hol Pharmacol 2011;24:1087-91.12. Happle R, Hausen BM, Wiesner Menzel L. Diphen-cyprone in the treatment of alopecia areata. Acta Derm Venereol (Stockh) 1983;63:49-52. 13. D’Ovidio R. Koebner phenomenon in alopecia areata: rapid appearance after Trichogram. Impli-cations for the pathogenesis and therapy. G Ital Dermatol Venereol 2013;148: 225-9.14. Schauber J, Gallo RL. The vitamin D pathway: a new target for control of the skin’s immune re-sponse? Exp Dermatol 2008;17: 633-9.15. D’Ovidio R, Vessio M, d’Ovidio F. Reduced level of25-hydroxyvitamin D in chronic/relapsing Alo-pecia Areata. Dermatondocrinol 2013;5:36-7.Ganzetti et al. Acta Dermatovenerol CroatOsteopontin in alopecia areata    2015;23(1):19-22

Osteopontin: A New Facilitating Factor in Alopecia Areata Pathogenesis?

Osteopontin (OPN) is a multifunctional glycophosphoprotein secreted by many cell types, including osteoblasts, lymphocites, macrophages, epithelial cells, and vascular smooth muscle cells. It has been implicated in many physiological and pathological processes, such as cell-mediated immunity, inflammation, cell survival, and tumor invasion and metastasis. Osteopontin has multiple emerging roles in cutaneous biology and pathology and OPN involvement has been emphasized in Th1-mediated diseases such as psoriasis. Alopecia areata (AA) is a form of non-scarring hair loss affecting anagen stage hair follicles with a multifactorial autoimmune pathogenesis characterized by a prevalent Th1 cytokine profile. Given the role of osteopontin in Th1-mediated inflammation, we have postulated that OPN may be involved in AA pathogenesis. The aim of our study was to investigate plasma OPN level in alopecia areata before and after DPCP treatment. Our results showed that OPN plasma levels in patients with alopecia areata were higher than in healthy controls, but patients achieving complete recovery after DPCP treatment did not show a statistically significant reduction of OPN plasma levels

Osteopontin: A New Facilitating Factor in Alopecia Areata Pathogenesis?

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