Serum Amyloid A Alters the Properties of Healthy Human Plasma Causing Cultured THP-1 Human Macrophages to Exhibit an Atherogenic Phenotype
Eric Lam1, 2, Lora J. Kasselman2, Heather A. Renna2, Daniel S. Glass2, Joshua DeLeon2, Steven E. Carsons2, Allison B. Reiss2. 1New York Institute of technology College of Osteopathic Medicine, Flushing, New York, United States, 2Medicine, NYU Winthrop Hospital, Mineola, New York, United States
Purpose of Study Rheumatoid Arthritis (RA) is associated with early and accelerated atherosclerosis leading to increased morbidity and mortality. Our group has demonstrated that exposure of THP-1 human macrophages to plasma from RA patients affects expression of proteins involved in lipid handling in an atheroma-promoting manner, leading to lipid overload and foam cell formation. Serum amyloid A (SAA) is both an acute-phase protein and an apolipoprotein released in response to inflammation. SAA is highly elevated in RA. This study evaluates the effect of adding SAA to healthy control (HC) plasma in order to determine whether exogenous SAA will re-create the atherogenic impact of RA plasma on macrophages.
Methods Used THP-1 differentiated macrophages (106/ml) were incubated for 18h in RPMI media in the presence of 5% plasma from RA or HC subjects (n=3 of each). Plasma was obtained under an NYU Winthrop IRB-approved protocol. SAA (0, 1 or 10 μg/ml) was added to HC plasma. Macrophage mRNA was extracted and reverse transcribed to cDNA. Cholesterol efflux proteins: ATP binding cassette transporter (ABC)A1, ABCG1 and 27-hydroxylase were quantified by real-time RT-PCR using specific primers for each gene and normalized to housekeeping gene GAPDH. One way ANOVAs and appropriate post-hoc tests were performed on normally distributed data. The significance level was set to 0.05.
Summary of Results The level of all 3 atheroprotective proteins was suppressed in RA versus HC plasma. Addition of SAA suppressed ABCA1, ABCG1 and 27-hydroxylase. 27-hydroxylase was reduced to a level below that found in RA plasma (p<0.01).
Conclusions Addition of SAA to HC plasma induces an RA-like inflammatory state. SAA in RA plasma may contribute to the atherogenic effect of this plasma by compromising cholesterol outflow from macrophages, leading to lipid engorgement. The ability of SAA to disable the critical reverse cholesterol transport pathway suggests a mechanism by which RA raises atherosclerotic risk. Novel therapies targeted to reduce SAA levels in RA may improve cholesterol efflux and cardiovascular risk profile.
Real-time RT-PCR quantitation of mRNA expression levels of ABCA1, ABCG1, and 27-hydroxylase in THP-1 macrophages after treatment with 5% plasma from RA subject, HC subject alone, HC subject with 1 mg/ml SAA, or HC subject with 10 mg/ml SAA.
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