0626: Elucidating the Expression and Role of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in Human Rheumatoid Arthritis Synovial Fibroblasts and a Rat Adjuvant-induced Arthritis Model

Meena Afroze Shanta¹, Farheen Sultan Shaikh², Bhanupriya Madarampalli³ and Salahuddin Ahmed², ¹College of Pharmacy and Pharmaceutical Science, Washington State University, Spokane, WA, ²Washington State University, Spokane, WA, ³University of Washington, Bellevue, WA

Background/Purpose: Proprotein convertase subtilisin/Kexin type-9 (PCSK9) is the most recognized serine protease for its pathological role in cardiovascular diseases, however, its role in inflammatory diseases, including rheumatoid arthritis (RA) remains unknown. Since cardiovascular comorbidities are common in RA patients, it is important to decipher the role of PCSK9 in RA pathogenesis.

Methods: The enzymatic activity of PCSK9 was determined using the peptidyl-MCA substrate-based fluorometric method in RA synovial fibroblasts (RASFs) isolated from de-identified RA synovial tissues (ST) under the IRB approved protocol and in the liver homogenates and serum of the rat adjuvant-induced arthritis (AIA) model of RA. The expression of PCSK9 and its correlation with low-density lipoprotein receptor (LDLR) expression in a rat AIA-model of inflammatory arthritis was examined via Western blot. The effect of IL-1β, IL-6, or TNF-α on the activity of PCSK9 was determined in cultured human RASFs and HepG2 cells. To understand the role of PCSK9 in cytokine-induced inflammation, we evaluated the effect of recombinant human PCSK9 (rhPCSK9) and SBC-110736, a known PCSK9 inhibitor, on IL-1β-induced IL-6 and IL-8 production and cyclooxygenase-2 (COX-2) expression in RASFs using ELISA and Western blot, respectively. All the experiments were carried out at least in triplicates and the statistical values of p< 0.05 were considered significant.

Results: The enzymatic activity of PCSK9 was significantly inhibited in human RASFs by ~15-35% (p< 0.05) and in HepG2 cells by ~25-50% when treated with IL-1β, TNF-α, or IL-6 (p< 0.05). The PCSK9 activity in the liver homogenates and serum of AIA rats showed a concerted decrease with the disease severity compared to the naïve group (p< 0.05). Furthermore, Western blot analysis of the AIA liver homogenates showed that the PCSK9 expression was suppressed by >50% , and the LDLR expression was enhanced by ~2-fold when compared to the naïve group (p< 0.05). Pretreatment of human RASFs with rhPCSK9 (0.2-2 µg/ml) significantly inhibited IL-1β-induced IL-6 and IL-8 production in a dose-dependent manner (p< 0.05 and p< 0.01). On the contrary, the pretreatment of human RASFs with PCSK9 inhibitor (SBC-110736) further enhanced IL-1β-induced IL-6 and IL-8 production and increased the cellular expression of COX-2, dose-dependently.

Conclusion: Our preliminary findings suggest that PCSK9 activity and expression decline in RA and may potentially play an important role in the loss of protection from cytokine-induced inflammation. Further studies examining the role of PCSK9 in clinical subjects will provide a clearer understanding on its association with RA.

Disclosures: M. Shanta, None; F. Shaikh, None; B. Madarampalli, None; S. Ahmed, None.