P2Y13 receptor regulates HDL metabolism and atherosclerosis in vivo

Abstract

High-density lipoprotein (HDL) is known to protect against atherosclerosis by promoting the reverse cholesterol transport. A new pathway for the regulation of HDL-cholesterol (HDL-c) removal involving F1-ATPase and P2Y13 receptor (P2Y13R) was described in vitro, and recently in mice. However, the physiological role of F1-ATPase/P2Y13R pathway in the modulation of vascular pathology i.e. in the development of atherosclerotic plaques is still unknown. We designed a specific novel agonist (CT1007900) of the P2Y13R that caused stimulation of bile acid secretion associated with an increased uptake of HDL-c in the liver after single dosing in mice. Repeated dose administration in mice, for 2 weeks, stimulated the apoA-I synthesis and formation of small HDL particles. Plasma samples from the agonist-treated mice had high efflux capacity for mobilization of cholesterol in vitro compared to placebo group. In apoE-/- mice this agonist induced a decrease of atherosclerotic plaques in aortas and carotids. The specificity of P2Y13R pathway in those mice was assessed using adenovirus encoding P2Y13R-shRNA. These results demonstrate that P2Y13R plays a pivotal role in the HDL metabolism and could also be a useful therapeutic agent to decrease atherosclerosis. In this study, the up-regulation of HDL-c metabolism via activation of the P2Y13R using agonists could promote reverse cholesterol transport and promote inhibition of atherosclerosis progression in mice.

Figure 1. Increase of HDL recycling following activation of P2Y13R pathway in mice.

C57Bl/6J mice (n = 10) were fasted for 2 h followed by single oral dose of P2Y13R agonist CT1007900 at 3, 30 or 300 µg/kg. Six hours later bile acid content (panel A) and bile cholesterol content (panel B) were evaluated using enzymatic kits. Grey bars represent the amount of bile acid or bile cholesterol per mouse; black bars represent the concentrations of bile acid and bile cholesterol into gallbladder. Panel C, the kinetic of bile acid mobilization in gallbladder induced by CT1007900 at 300 µg/kg (???) by oral gavage (single dosing) using C57Bl/6J mice (n = 5) was evaluated and compared to vehicle treated animals (○). *p<0.05, **p<0.01, ***p<0.0005. Bile acid content of liver (panel D) was evaluated using enzymatic kit. * p<0.05, **p<0.01. Plasma cholesterol (panel E) and plasma apoA-I (panel F) concentrations were determined at different time points after single oral dose of P2Y13R agonist at 100 µg/kg () and compared to vehicle treated animals (○). Values in pre-dose groups for plasma cholesterol vary from 0.85 to 0.95 g/L, and 1.1 to 1.3 g/L for plasma apoA-I. Panel G, C57Bl/6J mice (n = 5) were intravenously injected with [³H]-cholesterol-labelled mouse HDL (10 µCi/mouse) and CT1007900 (10 nmole/kg or 4 µg/kg). Radioactivity present in the liver was determined 2 hours later. **p<0.01. Panel H, C57Bl/6J mice (n = 5) were dosed (single dosing) with CT1007900 (100 µg/kg) and intravenously injected with [³H]-cholesterol-labelled mouse HDL (10 µCi/mouse). Feces from individual mouse were collected for 6 h and extracted for cholesterol (empty bars) and bile acid content (grey bars) and the radioactivity was determined by scintillation counting. *p<0.05.

Figure 2. Repeated dosing of P2Y13R agonist decreases HDL-C.

Plasma cholesterol (panel A), plasma apoA-I (panel B) and plasma lipoproteins (panel C) concentrations were determined after 2 weeks oral dose of P2Y13R agonist at 100 µg/kg (grey bars) and compared to vehicle treated animals (empty bars). Values for plasma apoA-I vary from 1.5 to 1.85 g/L. *p<0.05. Panel D, Concentration of liver apoA-I was determined by Western-blot quantification (n = 4 mouse/group) using imageJ software. 40 µg of total liver extract (vehicle or CT1007900 at 100 µg/kg) were separated on the same 12.5% SDS-PAGE and probed with goat anti-apoA-I antibody. *p<0.05. Panel E, the ratios of apoA-I/plasma cholesterol concentrations were determined and compared to their respective pre-dose values. Panel F, HDL from C57Bl/6J mouse plasma were separated according to the size of the different HDL particles using the Lipoprint system. The data were expressed as the percentage of difference for each HDL subpopulation set to the HDL population in the pre-dose animals. **p<0.01. Panel G, Determination of cholesterol efflux capacity of mouse plasma (1% v/v) using pre-loaded [³H]-cholesterol-oxLDL macrophages. The results are expressed as a percentage of cholesterol efflux corrected from pre-dose. Values for cholesterol efflux before correction from pre-dose vary from 12–15%. *p<0.05.

Figure 3. Effect of CT1007900 on atherosclerotic plaque progression in carotids of apoE^−/− mice.

Pannel A. ApoE^−/− mice (n = 7) were ligatured on the upper part of the left carotids. On the day of the surgery the animals were placed on a HCD and given an oral gavage of vehicle or increasing doses of compound CT1007900. Ligatured carotids were lipid extracted in 2∶1 chloroform/methanol and the concentrations of total cholesterol were measured by HPLC. **p<0.01. Panel B, C and D: longitudinal sections of apoE^−/− mice ligated carotids were analyzed by hematoxylin eosin staining, Oil Red O staining and CD-68 antibody staining respectively. *p<0.05. Panel E: Liver unesterified cholesterol determination. **p<0.01. Panel F. ApoE^−/− mice (n = 10) were infected with 5×10⁹ adenoviral particles coding empty vector (mock) or vector encoding P2Y13R shRNA, 3 days before the ligation of the left carotid. On the day of surgery the animals were placed on a Western diet and also given oral gavage of vehicle or compound CT1007900 at 100 µg/kg, once a day for 2 weeks. Ligatured carotids were lipid extracted in 2∶1 chloroform/methanol. The concentrations in total cholesterol were measured by HPLC. **p<0.01.

Figure 4. Effect of P2Y13R agonist on atherosclerotic plaque progression in aortas of apoE^−/− mice.

ApoE^−/− mice (n = 20) were placed on a Western diet for 8 weeks and after one month of diet, they were dosed for 4 weeks with vehicle or 100 µg/kg of compound CT1007900. Aortas (n = 10) were lipid extracted in 2∶1 chloroform/methanol. Panel A, the concentrations in total cholesterol were measured by HPLC and GC/MS and compared to parallel apoE^−/− mice (n = 10) on normal Chow diet as a baseline Panel B, quantification of ORO staining area (n = 10). Panel C, quantification of the intima/media ratio (n = 10). Panel D, anti-VCAM1 antibody staining quantification area (n = 10). Panel E, F4/80 antibody staining quantification area (n = 10). Panel F to M, typical example of staining of aorta slides used for the quantifications. Panel F and J, ORO staining; panel G and K hematoxylin eosin staining; panel H and L, VCAM-1 antibody staining; panel I and M, macrophage staining of transversal sections of apoE^−/− mice aortas. *p<0.05, ***p<0.001.