Objectives: 1- To characterize the serum molecular profile associated to the increased cardiovascular (CV) risk in Rheumatoid Arthritis (RA) patients. 2- To evaluate the in vivo and in vitro effects of biological drugs on the reestablishment of this altered molecular profile.

Methods: Serum samples of 280 RA patients and 100 healthy donors (HD) were studied. miRNomes were identified using next-generation sequencing miRNA assay (HTG EdgeSeq technology). The inflammatory profile, Netosis-derived products, and circulating biomolecules related to oxidative stress were quantified using commercial kits. The Cardiovascular Risk SCORE for RA patients was calculated following EULAR recommendations. Carotid intima-media thickness (CIMT) was evaluated as early atherosclerosis marker. The in vivo effects of biologic drugs such as Infliximab (IFX), Tocilizumab (TCZ) and Rituximab (RTX) were evaluated before and after 6 months of therapy in 45, 20 and 25 RA patients, respectively. Serum from RA patients with high and low CV risk scores -either before and after IFX, TCZ and RTX therapies-, were further added to HUVECs, monocytes, and neutrophils purified from HD, and activity profiles were evaluated.

Results: The miRNA whole transcriptome assay identified 104 circulating miRNAs altered in RA patients. Functional classification (IPA) established their involvement in inflammatory response, as well as in immunological and hematological diseases. Circulating biomolecules related to inflammation -interleukins, chemokines, adhesion molecules-, Netosis -cell-free nucleosomes, elastase and DNA- and oxidative stress -lipoperoxides, nitrated proteins, and total antioxidant capacity- were also found coordinately altered in the serum of RA patients. Multivariate analyses showed that levels of a number of those altered biomolecules and circulating microRNAs were predictors of a high Cardiovascular Risk SCORE and the presence of a pathologic CIMT in RA patients.

The in vivo treatments with IFX, TCZ and RTX for six months reduced disease activity and induced the re-establishment of normal levels in those altered biomolecules in RA patients. Mechanistic in vitro studies showed increased pro-inflammatory profiles of leukocytes subsets and HUVECSs after treatment with serum from high CV risk score-RA patients. These profiles were reversed by incubation with serum from those patients after biologic drugs treatment.

Conclusion: 1. Specific mediators of inflammation, oxidative damage and Netosis, along with the microRNAs modulating their expression, coordinately contribute to the higher CV risk score present in RA patients. 2. Biologic drugs such as IFX, TCZ and RTX, restore the normal levels of these altered biomolecules, reducing the CV risk in RA patients.

Acknowledgement: Funded by PI-0285-2017, ISCIII, PI18/00837 and RIER RD16/0012/0015 co-funded with FEDER

Disclosure of Interests: Carlos Perez-Sanchez: None declared, Nuria Barbarroja Puerto: None declared, Pérez Sánchez Laura: None declared, Patricia Ruiz-Limon: None declared, Sara Remuzgo Martinez: None declared, Alejandro Ibañez-Costa: None declared, Maria Luque-Tevar: None declared, Alejandra M. Patiño-Trives: None declared, Iván Arias de la Rosa: None declared, Maria del Carmen Abalos-Aguilera: None declared, Rafaela Ortega Castro: None declared, Alejandro Escudero Contreras: None declared, Raquel López-Mejías: None declared, M Ángeles Aguirre-Zamorano: None declared, Eduardo Collantes Estevez: None declared, Miguel A González-Gay Grant/research support from: Prof. MA Gonzalez-Gay received grants/research supports from Abbvie, MSD, Jansen and Roche., Speakers bureau: Consultation fees/participation in company sponsored speaker’s bureau from Pfizer, Lilly, Sobi, Celgene, Novartis, Roche and Sanofi., Chary Lopez-Pedrera: None declared