Citrullination Accompanies the Development of Carotid Atherosclerotic Plaques
Authors: Kanonykina Anastasia , Velikanova Elena , Markova Victoria , Bogdanov Leo , Shishkova Daria , Shabaev Amin , Sinitsky Maxim , Sinitskaya Anna , Poddubnyak Alyona , Lazebnaya Anastasia , Stepanov Alexander , Tyurina Arina , Lobov Arseniy , Zainullina Bozhana , Yuzhalin Arseniy , Kutikhin Anton 
Published: October 2024

Background:
Citrullination represents a post-translational modification primarily mediated
by peptidylarginine deiminase (PADI) 2 and 4 and resulting in the conversion of positively charged peptidylarginine
to neutrally charged peptidylcitrulline. Molecular consequences of citrullination include
the generation of neoepitopes which provoke the production of autoantibodies implicated in the development
of autoimmune diseases. As citrullination initiates, promotes, and is enhanced by aseptic inflammation
which plays a pivotal role in atherosclerosis, we proposed that citrullination might accompany
the development of atherosclerotic vascular disease.

Objective:
To investigate features and patterns of citrullination in atherosclerotic plaques.

objective:
To investigate features and patterns of citrullination in atherosclerotic plaques.

Methods:
We collected carotid atherosclerotic plaques (n = 14) and adjacent arterial segments (n = 14)
which were pairwise excised during the carotid endarterectomy. The tissues were examined employing
proteomic profiling (ultra-high performance liquid chromatography-tandem mass spectrometry analysis),
haematoxylin and eosin staining, Western blotting and immunofluorescence staining for peptidylcitrulline,
PADI2, and PADI4, and gene expression analysis. To better explore the mechanisms of citrullination
in the neointima, we have also stained excised plaques for the extracellular vesicle markers
(CD9 and CD81) and assessed co-localisation of PADI2 (a citrullination marker) with CD81 (an extracellular
vesicle marker). In order to study the systemic response to citrullination in an atherosclerotic
vascular disease setting, we measured the level of anti-citrullinated protein antibodies in the serum of
patients with ischaemic stroke and healthy volunteers.

Results:
Proteomic profiling found 213 plaque-specific and 111 intact arteria-specific proteins, as well
as 46 proteins and 13 proteins which have been respectively upregulated or downregulated in plaques
as compared with the adjacent intact segments. Among the top 20 upregulated proteins were atherogenic
apolipoprotein B-100, iron-associated protein haptoglobin, and matrix metal-loproteinase-9, together
indicating the advanced stage of plaque progression. In comparison with the intact arterial segments,
plaques demonstrated protein signatures of innate immune response and oxidative stress,
suggesting aseptic inflammation as a driver of atherosclerotic vascular disease. Both peptidylcitrulline
and PADI2 have been abundant in the neointima but negligible in tunica media; further, the levels of
peptidylcitrulline, PADI2, and PADI4 were elevated in plaque lysates in comparison with those from
adjacent arterial segments (p = 0.025, 0.025, and 0.010, respectively). Notably, PADI2 and peptidylcitrulline
were co-localised with the cells in the neointima and a considerable proportion of PADI2 was
co-localised with CD81-positive extracellular vesicles (p = 0.003). Albeit citrullinated histone H3 and
myeloperoxidase showed higher signal in the neointima than in tunica media (p = 0.048 and 0.023, respectively),
we did not observe any signs of neutrophil extracellular traps (e.g., unwound chromatin or
co-localisation of citrullinated histone H3 with neutrophil elastase) in the plaque tissue. Serum anti-citrullinated
protein antibodies were not elevated in patients with ischaemic stroke (p = 0.71), suggesting
that vascular citrullination likely does not trigger a generalised immune response.

Conclusions:
The development of carotid atherosclerosis is associated with citrullination, although it
represents a local rather than systemic phenomenon in this clinical scenario.