Abstract Body: Background: Cardiovascular disease (CVD) is the leading cause of mortality worldwide, largely driven by atherosclerosis resulting from arterial cholesterol accumulation. Reverse cholesterol transport (RCT) is an atheroprotective pathway lowering cholesterol burden, in which ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid efflux to apolipoprotein A1 (ApoA1), initiating nascent HDL formation. ABCA1 also promotes phosphatidylinositol 4,5-bisphosphate (PIP2) translocation to the plasma membrane, facilitating ApoA1 anchoring. However, the role of PIP2 in regulating ABCA1 expression and RCT remains incompletely understood.
Hypothesis: PIP2 regulates in vitro and in vivo RCT.
Methods and Approach: Wild-type (WT) or CRISPR-Cas9-generated knockout of PIP5K1α (PIP2 biosynthetic enzyme catalyzing conversion of PI4P to PIP2) cells were used. Transgenic mice (C57BL6/J background) were generated by transducing mice with recombinant adeno-associated virus 8 (rAAV8) expressing the 3X-FLAG-tagged Pip5k1a gene under a strong promoter (human eukaryotic translation elongation factor 1a). ABCA1 expression was assessed using western blotting and immunofluorescence. PIP2 levels were determined by MassELISA. RCT studies were performed in mice using tracer radiolabelled cholesterol.
Results: Cholesterol-loading induced ABCA1 expression in WT THP-1 and HepG2 cells, while PIP5K1α-KO cells were severely defective in inducing ABCA1 expression (~66% and ~68% reduction, respectively, N=3-4, mean±SD, *p<0.05 by unpaired t-test and one-way ANOVA). Consistently, immunofluorescence analysis demonstrated ~60% (n=30 cells) reduced ABCA1 expression in cholesterol-loaded PIP5K1α-KO THP-1 cells relative to WT controls. PIP5K1α-transgenic mice showed a marked increase in hepatic/intestinal ABCA1 protein (>2-3.5-fold increase, N=4-9, mean±SD, with **p<0.01 by unpaired t-test) and ABCA1 mRNA (*p<0.05 by t-test). PIP5K1α-transgenic mice showed higher RCT to plasma (an increase of 1.4-fold at 24 h, 2.1-fold at 48 h, and 3-fold at 72 h) and liver (2.8-fold increase) vs. mice transduced with stuffer alone.
Conclusion: These findings identify a direct role for PIP2 as a critical regulator of ABCA1 expression and RCT. Targeting PIP5K1α-mediated PIP2 modulation represents a novel therapeutic strategy to enhance cholesterol efflux and reduce CVD risk.