Citrullination within the Atherosclerotic Plaque: A New Potential Target for Anti-Citrullinated Protein Antibodies - ACR Meeting Abstracts

Abstract Number: 1690

Citrullination within the Atherosclerotic Plaque: A New Potential Target for Anti-Citrullinated Protein Antibodies

Jeremy Sokolove¹, Orr Share¹, Matthew Brennan¹, Lauren J. Lahey¹, Amy H. Kao², Eswar Krishnan³, Mary Chester M. Wasko⁴ and William H. Robinson⁵, ¹Medicine, VA Palo Alto Health Care System and Stanford University, Palo Alto, CA, ²Lupus Center of Excellence, Allegheny Singer Research Institute, Pittsburgh, PA, ³Medicine, Standford University, Palo Alto, CA, ⁴West Penn Allegheny Health System, Temple University School of Medicine, Pittsburgh, PA, ⁵Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA

Meeting: 2012 ACR/ARHP Annual Meeting

Keywords: anti-citrullinated protein/peptide antibodies (ACPA), atherosclerosis and rheumatoid arthritis, pathogenesis

Session Information

Title: Rheumatoid Arthritis - Human Etiology and Pathogenesis I: Early Pathogenesis of Rheumatoid Arthritis

Session Type: Abstract Submissions (ACR)

Background/Purpose: Patients with rheumatoid arthritis (RA) are at increased risk for cardiovascular disease due to accelerated atherosclerosis. This risk has mainly been limited to seropositive population as measured by the presence of rheumatoid factor (RF). We hypothesized that citrullination of proteins within the atherosclerotic plaque, including citrullinated fibrinogen (cFb), could be targeted by circulating anti-citrullinated protein antibodies (ACPA) thus forming stimulatory immune complexes which further propagate the progression of atherosclerosis.

Methods: We performed proteomic and immunohistochemical studies of atherosclerotic lesions to identify citrullinated proteins. Lysates were prepared from atherosclerotic segments of human aortic arch obtained at autopsy, subjected to 1 and 2-D PAGE, and probed by western blot for the presence of citrulline modified proteins and fibrinogen. Plaque lystates were immunoprecipitated with anti-fibrinogen antibody and subjected to immunoblot with anti-modified citrulline antibodies. Paraffin sections of several human coronary artery plaques were examined by immunohistochemistry for the presence of citrullinated proteins, PAD4 enzyme, and fibrinogen. Levels of anti-CCP2, anti-citrullinated vimentin (cVim), and anti-cFb were measured in a cohort of 135 women with RF+ RA (93% anti-CCP2+) of at least 2 years duration who were well characterized for the presence of atherosclerosis by electron beam CT scan for levels of coronary artery calcification, and multiple linear regression performed to assess the association of these antibodies with calcified plaque burden.

Results: 1 and 2-D western analysis demonstrated several citrulline modified proteins within plaque lysate and the presence of cFb was confirmed by re-probing of the western blot membrane for fibrinogen. Immunoprecipitation of plaque lysate with anti-fibrinogen showed strong citrullination demonstrated by anti-citrulline antibody staining and mass spectroscopy. Additionally, immunoprecipitation of plaque lysate with RA patient-derived IgG again identified citrullinated fibrinogen by mass spectroscopy. Immunohistochemistry demonstrated co-localization of (i) citrullinated proteins, (ii) PAD4, and (iii) fibrinogen within the coronary artery atherosclerotic plaque. Finally, levels of anti-cFb, anti-cVim, and anti-CCP2 were associated with the increased atherosclerosis as measured by coronary calcium score. In age adjusted models, ACPA titers accounted for approximately 35% of variance in total calcium score and for each standard deviation increase in level of each ACPA, there was an increase in coronary artery calcium score of 400-500 units (anti-cFb P=0.02, anti-cVim P<0.01, anti-CCP2 P=0.01).

Conclusion: Citrulline modified proteins including citrullinated fibrinogen are prevalent within the atherosclerotic plaque and levels of ACPA are associated with degree of atherosclerotic burden. This observation suggests that humeral targeting of citrullinated epitopes, specifically cFb, within the atherosclerotic plaque could provide a mechanism for accelerated atherosclerosis observed in patients with ACPA+ RA.

Disclosure:

J. Sokolove,
None;

O. Share,
None;

M. Brennan,
None;

L. J. Lahey,
None;

A. H. Kao,
None;

E. Krishnan,
None;

M. C. M. Wasko,
None;

W. H. Robinson,
None.

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