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Anti-annexin V autoantibodies and vascular abnormalities in systemic sclerosis: a longitudinal study

Abstract

Background:

Annexins are a group of conserved proteins which exert several regulatory functions on various cellular activities. Increased frequency and levels of antibodies against annexin V have already been observed in several autoimmune diseases including systemic sclerosis (SSc), but their role as a vascular biomarker is unknown. The aim of this study was to determine the serum levels and the dynamical behavior of anti-annexin V antibodies over a 24 months follow-up in patients with SSc.

Methods:

In this bicentric cross-sectional study, 70 patients with SSc were consecutively selected from March 2016 to April 2017. Demographic and clinical features, including the presence of active DUs, were collected. Serum anti-annexin V IgG and IgM antibodies were measured at baseline and after 6, 12 and 24 months of follow-up. Videocapillaroscopy was performed in all patients.

Results:

Among the 70 SSc patients included anti-annexin V IgG was found in 11 patients (15.7%) (range of 15.88–39.48 U/mL) and anti-annexin V IgM in 10 patients (14.3%) (range of 14.16-22.69 U/mL) at baseline. During follow-up, the number of patients who were positive for anti-annexin V IgG and IgM remained stable over 24 months. Among the patients with positive anti-annexin V IgG at baseline the frequency of patients with necrosis or amputation of extremities, forced vital capacity less than 70% and pulmonary arterial hypertension (PAH) was significantly higher than in patients with negative anti-annexin V IgG antibodies. Patients with anti-annexin V IgG had also a higher Raynaud's Condition Score and a higher Health Assessment Questionnaire Disability Index (HAQ-DI) than patients without these antibodies at baseline. Patients with positive anti-annexin V IgM at baseline presented a higher frequency of PAH, compared to those with negative anti-annexin V IgM at baseline.

Conclusions:

Anti-annexin V antibodies are stable and do not change their positivity during a 24 month follow-up in SSc patients. Anti-annexin V IgG was associated with more severe interstitial lung involvement and digital microangiopathy, and patients with anti-annexin V IgG or IgM had a higher occurrence of PAH indicating an association of these biomarker with more severe disease.

Keywords:
Systemic sclerosis; Anti-annexin V; Digital ulcers; Vasculopathy; Biomarkers

Introduction

Systemic sclerosis (SSc) is an autoimmune connective tissue disease, characterized by heterogeneous clinical presentation that affects the skin and several internal organs [11. Varga J, Abraham D. Systemic sclerosis: a prototypic multisystem fibrotic disorder. J Clin Invest. 2007;1 17(3):557-67.]. The pathogenesis of SSc includes interplay between vascular injury, abnormalities of the cellular and humoral immune systems and tissue fibrosis of the skin and internal organs such as lung, heart, and gastrointestinal tract [22. Geyer M, Müller-Ladner U. The pathogenesis of systemic sclerosis revisited. Clinic Rev Allerg Immunol. 2011;40:92-103., 33. Abraham DJ, Krieg T, Distler J, Distler O. Overview of pathogenesis of systemic sclerosis. Rheumatol. 2009;48:iii3-7. https://doi.org/10.1093/rheumatology/ken481.
https://doi.org/10.1093/rheumatology/ken...
]. Circulating antibodies, alteration of immune mediators, and mononuclear cell infiltration into affected organs indicates that immune system dysfunction is important in the disease pathogenesis [22. Geyer M, Müller-Ladner U. The pathogenesis of systemic sclerosis revisited. Clinic Rev Allerg Immunol. 2011;40:92-103., 33. Abraham DJ, Krieg T, Distler J, Distler O. Overview of pathogenesis of systemic sclerosis. Rheumatol. 2009;48:iii3-7. https://doi.org/10.1093/rheumatology/ken481.
https://doi.org/10.1093/rheumatology/ken...
]. Endothelial cell dysfunction is an early event in SSc and plays a role in the progression of vasculopathy and fibrosis [44. Manetti M, Guiducci S, Ibba-Manneschi L, Matucci-Cerinic M. Mechanisms in the loss of capillaries in systemic sclerosis: angiogenesis versus vasculogenesis. J Cell Mol Med. 2010;14:1241-54.].

In recent years, several efforts have been made on the identification of serum biomarkers associated with disease severity, and with specific clinical features such as peripheral vasculopathy in SSc [55. Matsushita T, Takehara K. An update on biomarker discovery and use in systemic sclerosis. Expert Rev Mol Diagn. 2017;17(9):823-33.88. Avouac J, Meune C, Ruiz B, Couraud PO, Uzan G, Boileau C, et al. Angiogenic biomarkers predict the occurrence of digital ulcers in systemic sclerosis. Ann Rheum Dis. 2012;71(3):394-9.].

Annexins are a group of conserved proteins which exert several regulatory functions on various cellular activities. Autoantibodies directed toward annexin I, II, V and XI have been reported in different autoimmune diseases, including autoimmune rheumatic diseases, but their role in immune response is controversial [99. Iaccarino L, Ghirardello A, Canova M, Zen M, Bettio S, Nalotto L, et al. Anti-annexins autoantibodies: their role as biomarkers of autoimmune diseases. Autoimm Rev. 2011;10:553-8.]. Annexin V belongs to a calcium-dependent phospholipid-binding protein family and exerts potent anticoagulant effects [99. Iaccarino L, Ghirardello A, Canova M, Zen M, Bettio S, Nalotto L, et al. Anti-annexins autoantibodies: their role as biomarkers of autoimmune diseases. Autoimm Rev. 2011;10:553-8.1111. Sugiura K, Muro Y. Anti-Annexin V antibodies and digital ischemia in patients with scleroderma. J Rheumatol. 1999;26(10):2168-72.]. Annexin V is highly expressed by vascular endothelial cells and is also involved in the regulation of apoptosis and protection against both excessive coagulation and inflammatory activities [1212. El Serougy IM, Shahin AA, Soliman DA, Akhnoukh AF, Mousa SM. Clinical significance of serum anti-annexin V antibodies in Egyptian patients with scleroderma. Egypt J Immunol. 2009;16(1):1-8.1414. Satoh A, Suzuki K, Takayma E, Kojima K, Hidaka T, Kawakami M, et al. Detection of anti-annexin V antibodies in patients with antiphospholipid syndrome and systemic lupus erythematosus. J Rheumatol. 1999;26:1715-20.]. Annexin V is also highly expressed by villous placental syncytiotrophoblast at the maternal-fetal interface and has been shown to play a thrombomodulatory role within the placental blood circulation [99. Iaccarino L, Ghirardello A, Canova M, Zen M, Bettio S, Nalotto L, et al. Anti-annexins autoantibodies: their role as biomarkers of autoimmune diseases. Autoimm Rev. 2011;10:553-8.].

Increased frequency and levels of antibodies against annexin V have already been observed in several auto-immune diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), anti-phospholipid syndrome (APS), and SSc [99. Iaccarino L, Ghirardello A, Canova M, Zen M, Bettio S, Nalotto L, et al. Anti-annexins autoantibodies: their role as biomarkers of autoimmune diseases. Autoimm Rev. 2011;10:553-8., 1414. Satoh A, Suzuki K, Takayma E, Kojima K, Hidaka T, Kawakami M, et al. Detection of anti-annexin V antibodies in patients with antiphospholipid syndrome and systemic lupus erythematosus. J Rheumatol. 1999;26:1715-20., 1515. Rodríguez-García MI, Fernández JA, Rodríguez A, Fernández MP, Gutierrez C, Torre-Alonso JC. Annexin V autoantibodies in rheumatoid arthritis. Ann Rheum Dis. 1996;55(12):895-900. https://doi.org/10.1136/ard.55.12.895.
https://doi.org/10.1136/ard.55.12.895...
]. Anti-annexin V antibodies have also been associated with thrombotic and vessel occlusive events, recurrent miscarriages and preeclampsia, especially in patients with systemic lupus erythematosus and APS [99. Iaccarino L, Ghirardello A, Canova M, Zen M, Bettio S, Nalotto L, et al. Anti-annexins autoantibodies: their role as biomarkers of autoimmune diseases. Autoimm Rev. 2011;10:553-8., 1414. Satoh A, Suzuki K, Takayma E, Kojima K, Hidaka T, Kawakami M, et al. Detection of anti-annexin V antibodies in patients with antiphospholipid syndrome and systemic lupus erythematosus. J Rheumatol. 1999;26:1715-20.].

In patients with SSc, it has been previously suggested that anti-annexin V antibodies could lead to a thrombogenic state due to interference with the effects of annexin V [99. Iaccarino L, Ghirardello A, Canova M, Zen M, Bettio S, Nalotto L, et al. Anti-annexins autoantibodies: their role as biomarkers of autoimmune diseases. Autoimm Rev. 2011;10:553-8., 1313. Reutelingsperger CPM, Van Heerde WL. Annexin V the regulator of phosphatidilserine-catalyzed inflammation and coagulation during apoptosis. Cell Mol Sci. 1997;53:527-32.]. Few studies have evaluated anti-annexin V antibodies in patients with SSc, most of them showing an association with digital ischemia, but the role of these antibodies in SSc is still unknown [1010. Habeeb RA, Mansour HE, Abdeldayem AM, Abo-shady RA, Hassan IA, Saafan NK, et al. Anti-annexin V antibodies: association with vascular involvement and disease outcome in patients with systemic sclerosis. Clin Med Insights Arthritis Musculoskelet Disord. 2010;3:15-23.1212. El Serougy IM, Shahin AA, Soliman DA, Akhnoukh AF, Mousa SM. Clinical significance of serum anti-annexin V antibodies in Egyptian patients with scleroderma. Egypt J Immunol. 2009;16(1):1-8.]. Thus, the aim of this study was to determine the serum levels and the dynamical behavior of anti-annexin V antibodies over a 24 months follow-up in patients with SSc. In addition, we evaluated the association of anti-annexin V antibodies with the severity of peripheral vasculopathy, digital ulcers occurrence and the microvascular changes observed in nailfold videocapillaroscopy during follow-up.

Patients and methods

Study design and patients

In this prospective observational cohort study, 70 patients with SSc attending the Rheumatology Division of the University Hospital Maria Aparecida Pedrossian of the Federal University of Mato Grosso do Sul (UFMS) and of the Federal University of São Paulo (UNIFESP) were consecutively selected from March 2016 to April 2017 (baseline data published in Advances in Rheumatology DOI: https://doi.org/10.1186/s42358-019-0057-9) [1616. Horimoto AMC, Souza AS, Rodrigues SH, Kayser C. Risk of digital ulcers occurrence in systemic sclerosis: a cross-sectional study. Advances in Rheumatology. 2019;59:14-20.]. All patients were evaluated in four visits: at baseline and after 6, 12 and 24 months of follow-up. Patients had to meet the ACR/EULAR 2013 classification criteria for SSc [1717. van den Hoogen F, Khanna D, Fransen J, Johnson SR, Baron M, Tyndall A, et al. 2013 classification criteria for systemic sclerosis: an american college of rheumatology/european league against rheumatism collaborative initiative. Arthritis Rheum. 2013;65(11):2737-47.]. All subjects signed informed consent approved by the institutional ethical review board of both institutions (Federal University of São Paulo: CAAE: 53429216.5.1001.5505 and Federal University of Mato Grosso do Sul: CAAE: 49087115.6.0000.0021). Patients with overlapping rheumatic autoimmune diseases, malignancies and active infectious diseases were excluded.

Clinical assessment

At baseline, data regarding demographic and clinical features were collected, including information about age, gender, Raynaud's phenomenon (RP) duration before diagnosis, disease duration (defined as the onset of the first non-Raynaud's symptom), and modified Rodnan Skin Score (mRSS). The presence of calcinosis, telangiectasias, arthritis, renal crisis and esophageal dysmotility was also collected from all subjects. Interstitial lung involvement was evaluated by means of pulmonary function tests and computed tomography. The presence of pulmonary arterial hypertension (PAH) was assessed according to current definitions using Doppler echocardiography and right-sided heart catheterization [1818. Hoeper MM, Bogaard HJ, Condliffe R, Frantz R, Khanna D, Kurzyna M, et al. Definitions and diagnosis of pulmonary hypertension. J Am Coll Cardiol. 2013;24:42-50.]. The modified Rodnan Skin Score was evaluated in all patients by the same physician as previously described [1919. Clements P, Lachenbruch P, Siebold J, White B, Weiner S, Martin R, et al. Inter and intraobserver variability of total skin thickness score (modified Rodnan TSS) in systemic sclerosis. J Rheumatol. 1995;22:1281-5.]. The SSc patients were also classified into diffuse cutaneous (dcSSc) or limited cutaneous (lcSSc) disease groups [2020. LeRoy EC, Black C, Fleischmajer R, Jablonska S, Krieg T, Medsger TA Jr, et al. Scleroderma (systemic sclerosis): classification, subsets and pathogenesis. J Rheumatol. 1988;15:202-5.]. Drug therapy data were collected from all individuals at baseline.

Longitudinal evaluation

The presence and number of active digital ulcers (DUs) were recorded in each visit. Active DUs were defined as a loss of epithelialization and tissues involving, to different degrees, the epidermis, the dermis, the subcutaneous tissue and sometimes also involving the bone. The presence of necrosis and the amputation of the extremities were also recorded [2121. Amanzi L, Braschi F, Fiori G, Galluccio F, Miniati I, Guiducci S, et al. Digital ulcers in scleroderma: staging, characteristics and sub-setting through observation of 1614 digital lesions. Rheumatology (Oxford). 2010;49:1374-82.]. Patients were also classified according to the presence of recurrent DUs defined by the presence of one or more DUs in at least two visits [2222. Matucci-Cerinic M, Krieg T, Guillevin L, Schwierin B, Rosenberg D, Cornelisse P, et al. Elucidating the burden of recurrent and chronic digital ulcers in systemic sclerosis: long-term results from the DUO registry. Ann Rheum Dis. 2016;75:1770-6.].

Patients were instructed to complete the Raynaud's Condition Score (RCS) during the week before each visit, in which the difficulty the patient experienced with RP in the prior 24 h was estimated on a 0-10 scale (0 = no difficulty; 10 = extreme difficulty) [2323. Merkel PA, Herlyn K, Martin RW, Anderson JJ, Mayes MD, Bell P, et al. Scleroderma clinical trials consortium. Measuring disease activity and functional status in patients with scleroderma and Raynaud's phenomenon. Arthritis Rheum. 2002;46:2410-20.]. The Health Assessment Questionnaire Disability Index (HAQ-DI) (score from 0 to 3) was also recorded [2424. Johnson SR, Hawker GA, Davis AM. The health assessment questionnaire disability index and scleroderma health assessment questionnaire in scleroderma trials: an evaluation of their measurement properties. Arthritis Rheum. 2005;53:256-62.].

Nailfold videocapillaroscopy (NVC)

Videocapillaroscopy was performed using an optical videocapillaroscopic probe under a 200× magnification lens at the 3 visits (Optilia Medical OP-120020, Sweden). The images were captured and stored for further analysis. The following variables were assessed: the number of capillaries/mm, the number of enlarged capillaries (apical diameter > 20 μm), the number of giant capillaries (apical diameter > 50 μm), and the number of microhemorrhages. For the assessment of capillary loss (avascular score), the normal range of nine capillaries/mm was adopted [2525. Smith V, Pizzorni C, De Keyser F, Decuman S, Van Praet JT, Deschepper E, et al. Reliability of the qualitative and semiquantitative nailfold videocapillaroscopy assessment in a systemic sclerosis cohort: a two-Centre study. Ann Rheum Dis. 2010;69:1092-6., 2626. Smith V, De Keyser F, Pizzorni C, Van Praet JT, Decuman S, Sulli A, Deschepper E, Cutolo M. Nailfold capillaroscopy for day-to-day clinical use: construction of a simple scoring modality as a clinical prognostic index for digital trophic lesions. Ann Rheum Dis. 2011;70(1):180-3.]. The average number for each capillaroscopic variable was calculated from the analysis of four consecutive fields (1 mm each) in eight digits, excluding the thumbs. The mean scores from the eight fingers were added, and the total value was divided by the number of fingers evaluated. For each parameter, a semiquantitative rating scale was adopted as previously described [2525. Smith V, Pizzorni C, De Keyser F, Decuman S, Van Praet JT, Deschepper E, et al. Reliability of the qualitative and semiquantitative nailfold videocapillaroscopy assessment in a systemic sclerosis cohort: a two-Centre study. Ann Rheum Dis. 2010;69:1092-6.].

Autoantibody measurements

Serum anti-annexin V IgG and IgM antibodies were measured at baseline and after 6, 12 and 24 months of follow-up. Peripheral venous blood (20 mL) was collected in dry tubes. Sera were frozen at −20°C until analysis. Anti-annexin V antibodies IgG and IgM levels were measured using enzyme-linked immunosorbent assay (ELISA) (ORGENTEC Diagnostika GmbH, Mainz, Germany), according to the manufacturers’ instructions. Samples below the cut-off value of 8 units/mL were considered negative. Samples ≥ than 8 units/mL were considered positive, in compliance with the manufacturer's recommendation. Anticentromere (ACA), anti-Scl-70 and anti-RNA polymerase III (RNAP III) levels were measured at baseline using enzyme-linked immunosorbent assay (ELISA) (QUANTA Lite Centromere CENP-A & CENP-B, QUANTA Lite TM Scl-70 and QUANTA Lite RNA Pol III, respectively, INOVA Diagnostics, San Diego, CA, USA), according to the manufacturers’ instructions. Samples above the cut-off value of 20 units/mL were considered positive.

ELISA was also used to measure anticardiolipin IgM/IgG (Sigma Laboratory, Darmstadt, Germany) and anti-beta 2 glycoprotein 1 (ORGENTEC Diagnostika GmbH, Mainz, Germany). Tests were considered positive if the titer was > 20.0 U/ml for IgG/IgM anticardiolipin and > 8.0 U/ml for IgG/IgM anti-beta 2 glycoprotein 1. Lupus anticoagulant was detected according to the recommendations of the International Society of Thrombosis and Hemostasis (ISTH) using tests to verify the prolongation of clotting assays, such as activated partial thromboplastin time (aPTT), kaolin clotting time, and dilute Russell viper venom time (DRVVT). Then, the presence of lupus anticoagulant was confirmed by mixing normal platelet-poor plasma with the patient's plasma.

Statistical analysis

The Kolmogorov-Smirnov test was used to evaluate normality distribution. Differences between two groups were analyzed by t-test or the Mann-Whitney test for continuous variables. The chi-squared test was used to analyze categorical variables. The Kruskal-Wallis test was used to evaluate differences between three groups. The Dunn's post-test was used to pinpoint which specific means are significant from the others. Generalized linear models (GLMs) and Bonferroni post hoc tests were carried out to compare differences among different times of assessment (baseline, 6 months, 12 months and 24 months) and between groups. Pearson's correlation coefficients were used to evaluate the correlation between variables. Statistical analysis was performed using SPSS statistical software, version 23.0 (SPSS Inc., Chicago, IL, USA). A p-value < 0.05 was considered significant [2727. Rowe P. Essential statistics for the pharmaceutical sciences. Chichester: Wiley; 2007.].

Results

Among the 70 patients included, most were women (92.9%), with a mean age of 46.8 ± 12.5 years, a mean RP duration before diagnosis of 5.10 ± 6.15 years, and a mean disease duration of 9.41 ± 6.26 years. Twenty-five patients (35.7%) had dcSSc, and 45 patients (64.3%) had lcSSc. Active DUs were observed in 14 patients and active DUs or gangrene and amputation of the extremities in 15 patients at baseline. Eighteen patients (25.7%) were currently using intravenous cyclophosphamide, 9 patients (12.9%) were using methotrexate, and 7 patients (10.0%) were using mycophenolate. Corticosteroids (prednisone < 10 mg/day) were used by 17 patients (24.3%), calcium channel blockers by 45 patients (64.3%), phosphodiesterase-5 inhibitors by 18 patients (25.7%), antagonists of endothelin-1 (bosentan) by 3 patients (4.3%), and rituximab by 6 patients (8.6%). During the observation period, medication use was stable for the drug classes recorded except for cyclophosphamide. The evaluation of the scleroderma-specific autoantibodies showed the presence of ACA in 28 patients (40%), anti-topo I in 21 patients (30%), and anti-RNAP III in 5 patients (7.1%) (Table 1).

Table 1
Demographic and clinical features of the SSc patients

The evaluation of the anti-annexin V autoantibodies showed the presence of anti-annexin V IgG in 11 patients (15.7%) (range of 15.88-39.48 U/mL) and antiannexin V IgM in 10 patients (14.3%) (range of 14.16-22.69 U/mL) at baseline. During follow-up, the number of patients who were positive for anti-annexin V IgG and IgM remained stable over 24 months (Table 2).

Table 2
Longitudinal data of anti-annexin V serum levels, severity of Raynaud's phenomenon, HAQ-DI and nailfold capillaroscopy of the 70 SSc patients

The frequency of patients with active DUs remained stable during 24 months (p = 0.638). Among the 70 patients, 12 (17.1%) had recurrent DUs during longitudinal follow-up. The RCS was significantly lower at 6 months than at 12 or 24 months (p = 0.015), and no difference was observed between the 6-month analysis and baseline. The mean number of DU and the HAQ-DI remained stable during follow-up. The evaluation of NVC showed a significant decrease in the number of capillaries/mm and microhemorrhages and an increase in the number of enlarged capillaries and giant capillaries and in the avascular score throughout the 24-month follow-up (Table 2).

The serum levels of anti-annexin V IgG and IgM antibodies were similar between patients with DUs and without DUs (Anti-annexin V IgG: 6.27 ± 6.05 U/mL versus 6.21 ± 6.78 U/mL, p = 0.975; Anti-annexin IgM: 4.43 ± 2.92 versus 6.25 ± 5.50 U/mL, p = 0.097, respectively) or in patients with active DUs or gangrene and amputation compared to those without these abnormalities at baseline (Anti-annexin V IgG: 6.04 ± 5.90 versus 6.27 ± 6.83 U/mL, p = 0.904; Anti-annexin V IgM: 4.38 ± 2.82 versus 6.29 ± 5.55 U/mL, p = 0.073, respectively).

Among the 11 patients with positive anti-annexin V IgG at baseline the frequency of patients with necrosis or amputation of extremities, forced vital capacity less than 70% and PAH was higher than in patients with negative anti-annexin V IgG antibodies. Patients with anti-annexin V IgG had also higher RCS and HAQ-DI scores than patients without these antibodies at baseline (Table 3). Patients with positive anti-annexin V IgM at baseline presented a higher frequency of PAH, compared to those with negative anti-annexin V IgM at baseline. The frequency of interstitial lung involvement on CT scan was of 72.7 and 70% in patients with anti-annexin V IgG and IgM antibodies compared to 47.5 and 48.3% in patients with negative anti-annexing V IgG and IgM antibodies, respectively. There was no significant difference in the frequency of active and recurrent digital ulcers, and other clinical variables between patients with positive or negative anti-annexin V IgG or IgM antibodies at baseline (Table 3).

Table 3
Clinical variables according to the presence or absence of anti-annexin V at baseline

Among the 13 patients with persistent positive antiannexin V IgG measurement, 09 (69.2%) had persistent positive measurements for this autoantibody. Among the 11 patients with persistent positive anti-annexin V IgM measurement, 07 (63.6%) had persistent positive measurement for this autoantibody during longitudinal follow-up. Interestingly, 5 patients were positive for both antibodies (anti-annexin V IgG and IgM) in all measurements.

There was no correlation between the serum levels of anti-annexin V IgG or IgM at baseline and age, RP duration or disease duration, scleroderma renal crisis or telangiectasias or the number of active DUs evaluated at different time points (data not shown). However, the RCS and HAQ-DI score were statistically higher in patients with positive anti-annexin V IgG antibodies compared to those with negative anti-annexin V IgG at all evaluations (data not shown).

Discussion

This is the first study that evaluated the frequency of anti-annexin V antibodies during a longitudinal follow-up in patients with SSc. Vascular involvement and endothelial dysfunction are primary events in SSc, resulting in vascular obliteration and decreased blood flow to the organs [2828. Silva I, Teixeira A, Oliveira J, Almeida I, Almeida R, Águas A, et al. Endothelial dysfunction and Nailfold Videocapillaroscopy pattern as predictors of digital ulcers in systemic sclerosis: a cohort study and review of the literature. Clinic Rev Allerg Immunol. 2015;49:240-52.]. The clinical expression of vasculopathy is extremely heterogeneous and may manifest simply by RP, as well as by clinical manifestations such as digital ulcers or gangrene [2929. Silva I, Teixeira A, Oliveira J, Almeida I, Almeida R, Vasconcelos C. Predictive value of vascular disease biomarkers for digital ulcers in systemic sclerosis patients. Clin Exp Rheumatol. 2015;33(4 Suppl 91):S127-30.] or even life-threatening severe manifestations such as pulmonary arterial hypertension [3030. Davie NJ, Schermuly RT, Weissmann N, Grimminger F, Ghofrani HA. The science of endothelin-1 and endothelin receptor antagonists in the management of pulmonary arterial hypertension: current understanding and future studies. Eur J Clin Investig. 2009;39(2):38-49. https://doi.org/10.1111/j.1365-2362.2009.02120.x.
https://doi.org/10.1111/j.1365-2362.2009...
]. In this context, there is a need to identify biomarkers that are easily applicable in clinical practice, to identify patients at risk of developing more severe manifestations or certain phenotypes [3131. Kayser C. The search for new biomarkers in systemic sclerosis. Rev Bras Reumatol. 2016;56:285-6.].

Thus, in the present study, the role of anti-annexin V IgM and IgG autoantibodies as a biomarker of peripheral microangiopathy or internal organ involvement was prospectively evaluated. In particular, the presence of active DUs, gangrene and amputation, and the microvascular changes evaluated by videocapillaroscopy, were evaluated during a 24 month follow-up.

In our study, anti-annexin V IgG antibodies were observed in 15.7% of patients and IgM class antibodies in 14.3% of patients at baseline. At baseline, patients with positive anti-annexin V IgG showed a higher frequency of gangrene and amputation of the extremities, a lower FCV and a higher frequency of PAH. Anti-annexin V IgG was also associated with a worse RCS and HAQ-DI.

Our results are in agreement with the results of Sugiura and Muro (1999), who found a frequency of anti-annexin V IgG positivity in 18.2% of SSc patients [1111. Sugiura K, Muro Y. Anti-Annexin V antibodies and digital ischemia in patients with scleroderma. J Rheumatol. 1999;26(10):2168-72.]. In addition, the authors found higher anti-annexin V titers in the group of patients with digital ischemia (ulcer of the fingertip or gangrene of the finger) compared to patients without digital ischemia (8.3 U/ml versus 2.7 U/ml, p <0.004) [1111. Sugiura K, Muro Y. Anti-Annexin V antibodies and digital ischemia in patients with scleroderma. J Rheumatol. 1999;26(10):2168-72.]. Although an association with anti-annexin V and active digital ulcers was not observed in our study, an association with more severe peripheral microangiopathy evaluated by means of gangrene and amputation of the extremities was found, suggesting that anti-annexin V could be a biomarker of more severe vasculopathy. Moreover, the lower FCV and a higher frequency of PAH in patients with anti-annexin V suggest a possible association of anti-annexin V with more severe disease. In accordance with these results, antiannexin V IgG-positive patients had worse quality of life indices, as measured by HAQ-DI.

Two other studies have evaluated anti-annexin V in patients with SSc. El Serougy et al. [1212. El Serougy IM, Shahin AA, Soliman DA, Akhnoukh AF, Mousa SM. Clinical significance of serum anti-annexin V antibodies in Egyptian patients with scleroderma. Egypt J Immunol. 2009;16(1):1-8.], observed significantly higher levels of anti-annexin V IgG antibodies in 40 Egyptian patients with SSc compared to healthy controls. They also found higher levels of anti-annexin V IgG in patients with digital ischemia (ulcers or gangrene) and, similar to our findings, higher levels in patients with pulmonary fibrosis. The latest study was performed by Habeeb et al. [1010. Habeeb RA, Mansour HE, Abdeldayem AM, Abo-shady RA, Hassan IA, Saafan NK, et al. Anti-annexin V antibodies: association with vascular involvement and disease outcome in patients with systemic sclerosis. Clin Med Insights Arthritis Musculoskelet Disord. 2010;3:15-23.], who evaluated 20 SSc patients and found a prevalence of positivity of these antibodies in 75% of patients. This discrepant value might be related to the population studied or to the method used for the anti-annexin V measurement. The authors [1010. Habeeb RA, Mansour HE, Abdeldayem AM, Abo-shady RA, Hassan IA, Saafan NK, et al. Anti-annexin V antibodies: association with vascular involvement and disease outcome in patients with systemic sclerosis. Clin Med Insights Arthritis Musculoskelet Disord. 2010;3:15-23.] used a French Zymutest anti-annexin V IgG ELISA kit, while in our and El Serougy's study [1212. El Serougy IM, Shahin AA, Soliman DA, Akhnoukh AF, Mousa SM. Clinical significance of serum anti-annexin V antibodies in Egyptian patients with scleroderma. Egypt J Immunol. 2009;16(1):1-8.] the same German Orgentec kit was used, which evaluated both IgG and IgM class anti-annexin V antibodies.

In accordance to previous studies [1010. Habeeb RA, Mansour HE, Abdeldayem AM, Abo-shady RA, Hassan IA, Saafan NK, et al. Anti-annexin V antibodies: association with vascular involvement and disease outcome in patients with systemic sclerosis. Clin Med Insights Arthritis Musculoskelet Disord. 2010;3:15-23.1212. El Serougy IM, Shahin AA, Soliman DA, Akhnoukh AF, Mousa SM. Clinical significance of serum anti-annexin V antibodies in Egyptian patients with scleroderma. Egypt J Immunol. 2009;16(1):1-8.], we did not find significant correlation between anti-annexin V serum antibodies and patients’ age, gender, disease duration or clinical cutaneous involvement.

Recently there has been an increased interest in the study of anti-annexin V antibodies, since its inclusion has been considered as part of the diagnostic criteria of patients with antiphospholipid syndrome [3232. Ogawa H, Zhao D, Dlott JS. Elevated anti-annexin V antibody levels in antiphospholipid syndrome and their involvement in antiphospholipid antibody specificities. Am J Clin Pathol. 2000;114:619-28.], particularly in those patients in which conventional autoantibodies are negative (anti cardiolipin, lupus anticoagulant and anti-beta 2 glycoprotein 1). Mekinian et al. [3333. Mekinian A, Bourrienne MC, Carbillon L, Benbara A, Noémie A, Chollet-Martin S, et al. Non-conventional antiphospholipid antibodies in patients with clinical obstetrical APS: prevalence and treatment efficacy in pregnancies. Semin Arthritis Rheum. 2016;46(2):232-7.] found that 68% of patients with clinical criteria for obstetrical antiphospholipid syndrome (APS) that were seronegative for conventional antiphospholipid antibodies (APL) have non-conventional APL, mostly represented by anti-annexin V IgG antibodies. As expected, in our study, the frequencies of anticardiolipin, anti-beta 2 glycoprotein 1 or lupus anticoagulant antibodies were low, not allowing an analysis between these antibodies and anti-annexin V. In other autoimmune diseases, anti-annexin V was found in 3.8% of patients with SLE with no clinical or serological features of APS, 28.0% of patients with SLE having only serological signs of APS, and 30.4% of patients with clinical symptoms and serological signs of APS [99. Iaccarino L, Ghirardello A, Canova M, Zen M, Bettio S, Nalotto L, et al. Anti-annexins autoantibodies: their role as biomarkers of autoimmune diseases. Autoimm Rev. 2011;10:553-8.].

During the 24 months follow-up, anti-annexin V IgG and IgM antibodies showed no statistically significant variations, indicating that longitudinal measurement of anti-annexin V are not useful in the clinical practice. In general, when the patient was positive for an anti-annexin V antibody, he was always positive throughout the 24 months of clinical observation, without large variations in the titers of these autoantibodies. The number of patients with active DUs, gangrene and amputation and the mean number of DUs also remained stable during follow-up.

The study of microvascular abnormalities evaluated with videocapillaroscopy was also performed prospectively. In agreement with previous studies [3434. Cutolo M, Herrick AL, Distler O, Becker MO, Beltran E, Carpentier P, et al. Nailfold Videocapillaroscopic features and other clinical risk factors for digital ulcers in systemic sclerosis. Arthritis Rheum. 2016;68:2527-39.3636. Sebastiani M, Manfredi A, Vukatana G, et al. Predictive role of capillaroscopic skin ulcer risk index in systemic sclerosis: a multicentre validation study. Ann Rheum Dis. 2012;71:67-70.], a worsening of microvascular abnormalities was observed during follow-up. No worse videocapillaroscopy was observed in patients with positive anti-annexin V antibodies.

In relation to other vascular changes, patients with positive IgG and IgM anti-annexin V showed a higher frequency of PAH. Previous studies reinforce that anti-annexin V autoantibodies could play a role in pathogenesis of SSc. By binding to vascular endothelial cells, they would promote apoptosis and cytokine release that could contribute to pulmonary vasculopathy and fibrosis [99. Iaccarino L, Ghirardello A, Canova M, Zen M, Bettio S, Nalotto L, et al. Anti-annexins autoantibodies: their role as biomarkers of autoimmune diseases. Autoimm Rev. 2011;10:553-8.]. Indeed, in our study, a higher number of patients with anti-annexin V class IgG had FVC < 70% compared to patients without these antibodies. Although not statistical significant, interstitial lung involvement on CT scan was observed in higher frequency of patients with positive anti-annexin V antibodies. Thus, the present results suggest a possible association of anti-annexin V antibodies and the presence of PAH and pulmonary fibrosis.

Our study has some limitations including the lack of organ involvement evaluation during follow-up and the lack of a healthy control group.

In summary, we found that anti-annexin V antibodies remained stable during follow-up in patients with SSc. SSc patient's with positive anti-annexin V antibodies had worse digital microangiopathy and a higher frequency of interstitial lung involvement and PAH, suggesting that anti-annexin V antibodies could play a role in the pathogenesis of SSc patients as well as be associated with more severe disease.

Conclusion

In conclusion, in this 24-months prospective study, the presence of anti-annexin V IgG was associated with more severe interstitial lung involvement and digital microangiopathy, and the presence of anti-annexin V antibodies, either of the IgG or IgM class, with a higher occurrence of PAH. Prospective analyses are needed to confirm the value of these variables in predicting the occurrence of these manifestations in SSc patients.

  • Authors’ information
    Prior related work:
    1. HORIMOTO, AMC; NAKA, EN; COSTA, MR; TAKAHASHI, F; REZENDE, MC; BARRIOS, L; PRADEBON, EP; FINOTTI, LT; FERRA, FKM; RONDON, RMR; MACH ADO, NP; TAVARES, FMAA; ALVES, TP; OVIDIO, RA; COSTA, IP. Incidence and prevalence of systemic sclerosis in Campo Grande, State of Mato Grosso do Sul, Brazil. Revista Brasileira de Reumatologia (English Edition), p. 107-114, 2016.

    2. ANDRIGUETI, FV; ARISMENDI, MI; EBBING, PCC; KAYSER, C. Evaluation of the effect of sildenafil on the microvascular blood flow in patients with systemic sclerosis: a randomized, double-blind, placebo-controlled study. CLINICAL AND EXPERIMENTAL RHEUMATOLOGY, v. 35, p. 151-158, 2017.

    3. ARAÚJO, FC; CAMARGO, CZ; KAYSER, C. Validation of the ACR/EULAR classification criteria for systemic sclerosis in patients with early scleroderma. RHEUMATOLOGY INTERNATIONAL, v. 37, p. 1825-1833, 2017.

    4. PIOTTO, DGP; SEKIYAMA, JY; KAYSER, C; YAMADA, M; LEN, C; TERRERI, MTRA. Nailfold videocapillaroscopy in healthy children and adolescents: description of normal patterns. Clinical and Experimental Rheumatology, v. 34, p. 193-199, 2016.

    5. SOUZA, EJR; MULLER, CS; HORIMOTO, AMC; REZENDE, RA; GUIMARÃES, I; MARIZ, HA; DANTAS, AT; DA COSTA, IP; DEL-RIO, APT; SEKIYAMA, J; KAHWAGE, CB; KAYSER, C. Geographic variation as a risk factor for digital ulcers in systemic sclerosis patients: a multicentre registry. SCANDINAVIAN JOURNAL OF RHEUMATOLOGY, v. 46, p. 1-8, 2016.

    6. ANDRIGUETI, FV; ARISMENDI, MI; EBBING, PCC; KAYSER, C. Decreased numbers of endothelial progenitor cells in patients in the early stages of systemic sclerosis. Microvascular Research (Print), v. 98, p. 82-87, 2015.

    7. MINIER, T. GUIDUCCI, S. BELLANDO-RANDONE, S. BRUNI, C. LEPRI, G. CZIRJAK, L. DISTLER, O. WALKER, U. A. FRANSEN, J. ALLANORE, Y. DENTON, C. CUTOLO, M. TYNDALL, A. MULLER-LADNER, U. MATUCCICERINIC, M. AIRO, P. ZINGARELLI, S. ANANIEVA, L. DESINOVA, O. ANCUTA, C. M. BELIBOU, C. I. AVOUAC, J. BECVAR, R. SKACELOVA, S. BERETTA, L., VIGONE, B. CARAMASCHI, P. SABBAGH, D. CARPENTIER, P. DAMJANOV, N. SIMIC-PASALIC, K. DISTLER, J. H. FARGE-BANCEL, D. HADJKHELIFA, S. FOTI, R. DI GANGI, M. DE LA PENA LEFEBVRE, P. G. HACHULLA, E. SALVADOR, M. J. KAYSER, C. CAMARGO, C. Z. KUMANOVICS, G. LI, M. XU, D. MARASINI, B. BELLOLI, L. MAURER, B. MAYER, M. MIHAI, C. GHERGHE, A. M. RICCIERI, V. STEFANANTONI, K. SALSANO, F. ROSATO, E. SENECAL, J.-L. KOENIG, M. SENET, P. FRANCES, C. SIPEK, A. STAN KOVIC, A. STAMENKOVIC, B. SMITH, V. TARNER, I. H. WILAND, P.; Preliminary analysis of the Very Early Diagnosis of Systemic Sclerosis (VEDOSS) EUSTAR multicentre study: evidence for puffy fingers as a pivotal sign for suspicion of systemic sclerosis. Annals of the Rheumatic Diseases, v. 73, p. 2087-2093, 2014.

    8. CAMARGO, CZ; SEKIYAMA, JY; ARISMENDI, MI; KAYSER, C. Microvascular abnormalities in patients with early systemic sclerosis: less severe morphological changes than in patients with definite disease. Scandinavian Journal of Rheumatology (Trykt Utg.), v. 44, p. 1-8, 2014.

    9. HORIMOTO, AMC; SOUZA, AS; RODRIGUES, SH; KAYSER, C. Risk of digital ulcers occurrence in systemic sclerosis: a cross-sectional study. Adv. Rheumatol. 2019, v. 59, 14.

  • Funding
    The authors declare that they have used only private funds for research. development.
  • Availability of data and materials
    All data generated or analyzed during this study are included in this. published article, and its supplementary information files.
    The datasets generated and/or analyzed during the current study are not. publicly available due to ethics policy of the institutions but are available. from the corresponding author on reasonable request.
  • Ethics approval and consent to participate
    The manuscript was approved by the institutional ethical review board by. both institutions, in their respective ethics committees:
    Federal University of São Paulo number: 1.433.963.
    CAAE: 53429216.5.1001.5505.
    Federal University of Mato Grosso do Sul: 1.300.296.
    CAAE: 49087115.6.0000.0021.
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Acknowledgements

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Publication Dates

  • Publication in this collection
    07 Aug 2020
  • Date of issue
    2020

History

  • Received
    11 May 2020
  • Accepted
    20 July 2020
  • Published
    31 July 2020
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