P2Y13 receptor regulates HDL metabolism and atherosclerosis in vivo.

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

HDL and CER-001 Inverse-Dose Dependent Inhibition of Atherosclerotic Plaque Formation in apoE^-/- Mice: Evidence of ABCA1 Down-Regulation

<div><p>Objective</p><p>CER-001 is a novel engineered HDL-mimetic comprised of recombinant human apoA-I and charged phospholipids that was designed to mimic the beneficial properties of nascent pre-ß HDL. In this study, we have evaluated the dose-dependent regulation of ABCA1 expression <i>in vitro</i> and <i>in vivo</i> in the presence of CER-001 and native HDL (HDL3).</p><p>Methods and Results</p><p>CER-001 induced cholesterol efflux from J774 macrophages in a dose-dependent manner similar to natural HDL. A strong down-regulation of the ATP-binding cassette A1 (ABCA1) transporter mRNA (- 50%) as well as the ABCA1 membrane protein expression (- 50%) was observed at higher doses of CER-001 and HDL₃ compared to non-lipidated apoA-I. <i>In vivo</i>, in an apoE^-/- mouse “flow cessation model,” in which the left carotid artery was ligatured to induce local inflammation, the inhibition of atherosclerotic plaque burden progression in response to a dose-range of every-other-day CER-001 or HDL in the presence of a high-fat diet for two weeks was assessed. We observed a U-shaped dose-response curve: inhibition of the plaque total cholesterol content increased with increasing doses of CER-001 or HDL3 up to a maximum inhibition (- 51%) at 5 mg/kg; however, as the dose was increased above this threshold, a progressively less pronounced inhibition of progression was observed, reaching a complete absence of inhibition of progression at doses of 20 mg/kg and over. ABCA1 protein expression in the same atherosclerotic plaque was decreased by-45% and-68% at 50 mg/kg for CER-001 and HDL respectively. Conversely, a-12% and 0% decrease in ABCA1 protein expression was observed at the 5 mg/kg dose for CER-001 and HDL respectively.</p><p>Conclusions</p><p>These data demonstrate that high doses of HDL and CER-001 are less effective at slowing progression of atherosclerotic plaque in apoE^-/- mice compared to lower doses, following a U-shaped dose-response curve. A potential mechanism for this phenomenon is supported by the observation that high doses of HDL and CER-001 induce a rapid and strong down-regulation of ABCA1 both <i>in vitro</i> and <i>in vivo</i>. In conclusion, maximally efficient HDL- or CER-001-mediated cholesterol removal from atherosclerotic plaque is achieved by maximizing macrophage-mediated efflux from the plaque while minimizing dose-dependent down-regulation of ABCA1 expression. These observations may help define the optimal dose of HDL mimetics for testing in clinical trials of atherosclerotic burden regression.</p></div

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

<p>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.</p

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

<p>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.</p

Repeated dosing of P2Y13R agonist decreases HDL-C.

<p>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.</p

ABCA1 mRNA and protein decrease in J774 macrophages following CER-001 and HDL3 incubation.

<p>Panel A; ABCA1 mRNA level in J774 macrophages was determined in presence of different concentrations of CER-001, HDL3 or delipidated apoA-I for 24h. Panel B; Kinetic of ABCA1 mRNA level at a fixed concentration (250μg/mL) of CER-001, HDL3 or delipidated apoA-I. The qPCR data represent the means of triplicate determinations from a single experiment that is representative of three such experiments. Panel C; J774 macrophages were treated for 24 h in presence of CER-001, HDL3 or delipidated apoA-I at 250μg/mL (time 0). Compounds were removed and replaced with fresh medium for 24 h (time 24) and ABCA1 mRNA level was determined by qPCR. Panel D; The relative membrane protein expression of ABCA1 was measured by Western blot analysis for macrophages treated for 6h with CER-001, HDL3 or apoA-I at 250 μg/ml. cAMP was used for 24h at 300μM. Membrane protein loading for each sample was verified by Western blot using rabbit anti-Calnexin (1/1000 dilution). Membrane fractions were obtained after ultracentrifugation as described in Material & Method section. The data represent the means of triplicate determinations from a single experiment. * p>0.05.</p

cAMP stimulated-ABCA1 specific efflux decrease in J774 macrophages following CER-001 and HDL3 incubation.

<p>J774 macrophages were incubated with cAMP to increase the ABCA1 expression and then CER-001 and HDL3 at 25 μg/ml were added in the cell culture medium and the specific cAMP cholesterol efflux was determined. apoA-I (25 μg/ml) was used as reference for specific ABCA1-cholesterol efflux in the experiment. ** p<0.01, ***p<0.001.</p

Effect of dose-response of CER-001 and HDL3 in atherosclerotic plaque progression in ligatured carotid of apoE^-/- mice.

<p>The left carotid of apoE^-/- mice (n = 12) was ligatured, fed with HCD and treated (retro-orbital injection) every second day with different concentrations of CER-001 or HDL3 (8 infusions). The carotids were lipid extracted and cholesterol concentrations were determined by HPLC. Panel A; unesterified cholesterol. Panel B; total cholesterol. Panel C; protein level of ABCA1 was measured in the ligatured carotids using Western blot analysis as described in material and methods section. The data represent the means of ABCA1 expression from at least 5 different carotids. Representative Western-blot for carotid analysis was resolved with anti-ABCA1 antibody (1/1000 dilution) and anti-Calnexin antibody (1/1000). * p<0.05, ** p<0.01, ***p<0.005, ****p<0.001, *****p<0.0001</p