Liver-specific deletion of the PPAP2B gene worsens atherosclerosis in apoE-/- mice

Lipid phosphate phosphatases (LPPs) are integral membrane proteins that catalyze the dephosphorylation of a broad panel of lipid substrates. The LPP family is composed by three enzymes in mammals: LPP1, LPP2 and LPP3, coded by three independent genes named PPAP2A, PPAP2C and PPAP2B, respectively. The expression of LPP2 mRNA is found mainly in brain, pancreas and placenta, whereas LPP1 and LPP3 mRNAs appear quite ubiquitous. A great interest around PPAP2B/LPP3 was recently raised by the results of genome-wide association studies (GWAS) that identified PPAP2B as a gene playing a role in coronary artery disease (CAD) susceptibility. Aim of the study was to investigate the effect of Ppap2b deletion on atherosclerosis development in an athero-prone mouse model. Since constitutive deletion of Ppap2b in mice leads to embryo-lethality, conditional Ppap2b hepatocyte-specific null mice were generated by crossing floxed Ppap2b mice (Ppap2bf/f) with animals expressing the Cre recombinase under the control of the Albumin promoter. These mice were then crossed with apoE-/- mice, obtaining Ppap2bf/fApoE-/-Alb-Cre+ mice and Ppap2bf/fApoE-/-Alb-Cremice as controls. The liver was chosen as target organ of Ppap2b deletion, being the main source of circulating plasma lipids and lipoproteins, which importantly contribute to the atherosclerosis process. Mice of both genotypes were fed chow or Western diet for 40 and 12 weeks, respectively. At the end of the dietary treatments, atherosclerosis development was evaluated and plasma lipidomic analysis was performed. Compared with Cre recombinase negative mice, Ppap2b mRNA expression dropped over five-folds in the liver of Ppap2bf/fApoE-/-Alb-Cre+ mice. The presence of a residual Ppap2b functional gene in liver could be explained by the presence of non-hepatocyte cell types, such as sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells and lymphocytes. Comparable expression levels were observed in all the other organs/tissues assayed in Ppap2bf/fApoE-/-Alb-Cre+ and Ppap2bf/fApoE-/-Alb-Cre- animals, demonstrating the high specificity of Albumin promoter expression and Cre recombinase activity. Hepatocyte-specific Ppap2b deletion was associated to an increased atherosclerosis progression: this result was observed both at the aortic sinus (4.8±1.5×105 μm2 in Cre+ mice vs. 3.6±1.4×105 μm2 in Cre- mice), and at the aorta (Arch: 45.84±5.92% in Cre+ mice vs. 24.59±6.3% in Cre- mice. Thoracic: 3.59±1.79% in Cre+ mice vs.0.56±0.57% in Cre- mice. Abdominal: 4.37±2.25% in Cre+ vs. 4±4.37% in Cre- mice), when mice were fed Western diet, whereas no difference was found for atherosclerosis development between Ppap2bf/fApoE-/-Alb-Cre+ and Ppap2bf/fApoE-/-Alb-Cre- mice, when fed chow diet. With the aim of better understanding the impact of hepatic Ppap2b deletion on circulating lipids, possibly affecting atherosclerosis development, a plasma lipidomic analysis was conducted. When comparing Ppap2bf/fApoE-/-Alb-Cre+ and Ppap2bf/fApoE-/-Alb-Cre- mice fed chow diet, a significant increase of lactosylceramide in Ppap2bf/fApoE-/-Alb-Cre+ mice was observed. Western type diet, in both mouse lines, led to higher plasma concentrations of cholestery lesters, sphingolipids and most glycerophospholipids compared with the chow diet. Interestingly, in Ppap2bf/fApoE-/-Alb-Cre+ mice only, an elevation of lysophosphatidic acid and triglycerides was observed. Lipidomic comparison between Ppap2bf/fApoE-/-Alb-Cre+ and Ppap2bf/fApoE-/-Alb-Cre- mice after Western diet feeding showed significant differences between the two mouse lines. Specifically, in Ppap2bf/fApoE-/-Alb-Cre+ mice, plasma levels of lipids playing a role in atherosclerosis development such as lactosylceramide, lysophosphatidic acid, globotriosylceramide and lysophosphatidylinositol were significantly increased, compared with Western-fed Ppap2bf/fApoE-/-Alb-Cre- mice. In conclusion, the present work demonstrates for the first time, in an animal model, the role of Ppap2b/LPP3 in atherosclerosis development and provides an experimental evidence for the clinical observation relating PPAP2B polymorphisms to CAD susceptibility. Additionally, the study indicates that hepatic Ppap2b deletion leads to alterations in the plasma levels of several minor lipid species, whose role in atherosclerosis has been proven, providing a molecular basis for the observed results.