The role of Resolvin D1 and the G protein-coupled receptor 32 in Atherosclerosis
Final Report Abstract
Cardiovascular disease (CVD) is strongly linked with chronic inflammation and lack of resolution. By investigating the role of the pro-resolving lipid mediator Resolvin D1 (RvD1) and its receptor GPR32 in mouse models and human atherosclerosis, the project aimed to identify a potential benefit of RvD1 signaling in atherosclerosis. At first, several obstacles were met in the in vitro studies: the low expression in many cell types, the impact of the RvD1 solvent ethanol in all assays, and the fact that the electric cell impedance sensing system was not suitable for studying GPR32. A HEK293 cell line with stable GPR32 expression was generated, but it was not possible to measure the activity in functional assays. Only human macrophages could be stimulated to increased phagocytosis with RvD1. We moved forward and generated the GPR32 transgenic mouse in order to study this receptor directly in vivo. To save time, the GPR32 transgene was introduced in hypercholesteremic (ApoE knockout) mice that also lacked the endogenous murine receptor for RvD1 (FPR2 knockout) by random transgenesis. The GPR32myc(tg) x FPR2-/- x ApoE-/- mice were bred several generations with no observed adverse health effects. They presented however a significant lower weight as well as a dark brown fur color compared to the bigger black non-transgenic littermates. The insertion of the transgene was located on chromosome 2 and yielded a robust GPR32 mRNA expression in all tested organs. Following this characterization of the new mouse strain, the atherosclerosis disease model was started by placing the transgenic mice on a high fat diet (HFD) at 12 weeks of age. Plaque was confirmed in the aorta of females after 12 weeks of HFD and two groups were added that continued HFD for another four weeks including treatment with the GPR32 agonist RvD1 or Ethanol as a control administered by osmotic pumps implanted under the skin of the animals. At the 12 week HFD baseline, transgenic mice presented 30% less atherosclerotic plaque in the aortic arch. No changes in blood parameters, or triglyceride and cholesterol plasma levels was found. Although the transgenic mice weighed significantly less, no correlation between weight and plaque size could be detected for any of the mouse groups. Ongoing analyzes are looking at differences in the cell composition and collagen content of the aortic plaque. After four weeks of RvD1 treatment, the data hints at a further decrease in plaque, however not all experimental animals have been collected and analyzed yet. As an additional project, ApoE-/- mice with endogenous expression of FPR2 receptor underwent the same protocol. This approach made it possible to separately study effects of RvD1 on atherosclerosis mediated by the receptors GPR32 and FPR2. The data analysis is still ongoing for this experiment. In another approach, peritonitis was used as another model for resolution of inflammation. There was a significant difference in the macrophage to neutrophil ratio, presenting a higher increase of macrophages in the transgenic animals after 48 hours. This was followed by a tendency towards increased efferocytosis and significantly less TNFa expression in the GPR32 transgenic mice. When studying GPR32 expression in human carotid arteries, patients undergoing endarectomy had significantly less GPR32 compared to healthy vessels. In addition to macrophage markers, also FoxP3 regulatory T cells had a strong association with GPR32. Ongoing studies are further exploring this finding in mouse and human tissue. Two different studies were performed with saliva samples obtained from an Arterial Stiffness study cohort from our collaborators in Nancy, France. Firstly, salivary RvD1 to Leukotriene B4 ratio could be used as a biomarker for intima media thickness, a subclinical sign of atherosclerosis. Secondly, salivary phosphate and potassium were associated with cardiovascular risk. Both study showed the value of saliva as a possible source for biomarker studies and non-invasive diagnosis. Altogether, these findings support our hypothesis that RvD1 signaling through the GPR32 receptor is beneficial in atherosclerosis, leading to a less inflammatory phenotype and less plaque burden in the GPR32 transgenic mice. Furthermore, salivary RvD1 has the potential as a biomarker for non-resolving inflammation. Therefore, the RvD1 pathway presents a promising target for drug development in CVD.
Publications
- (2018) Resolution of Inflammation Through the Lipoxin and ALX/FPR2 Receptor Pathway Protects Against Abdominal Aortic Aneurysms. JACC. Basic to translational science 3 (6) 719–727
Petri, Marcelo H.; Thul, Silke; Andonova, Teodora; Lindquist-Liljeqvist, Moritz; Jin, Hong; Skenteris, Nikolaos-Taxiarchis; Arnardottir, Hildur; Maegdefessel, Lars; Caidahl, Kenneth; Perretti, Mauro; Roy, Joy; Bäck, Magnus
(See online at https://doi.org/10.1016/j.jacbts.2018.08.005) - Differential regulation of monocytic expression of leukotriene and lipoxin receptors. Prostaglandins Other Lipid Mediat. 2015 Sep;121(Pt A):138-43
Petri M, Thul S, Ovchinnikova O, Bäck M
(See online at https://doi.org/10.1016/j.prostaglandins.2015.07.005) - Lipoxygenases and Cardiovascular Disease. In: Lipoxygenases in Inflammation, 2016 Editor: Dieter Steinhilber; Springer
Laguna-Fernandez A, Petri M, Thul S, Bäck M
(See online at https://doi.org/10.1007/978-3-319-27766-0_6) - Low salivary resolvin D1 to leukotriene B4 ratio predicts carotid intima media thickness: A novel biomarker of non-resolving vascular inflammation. Eur J Prev Cardiol. 2017 Jun;24(9):903-906
Thul S, Labat C, Temmar M, Benetos A, Bäck M
(See online at https://doi.org/10.1177/2047487317694464) - ERV1/ChemR23 Signaling Protects from Atherosclerosis by Modifying oxLDL Uptake and Phagocytosis in Macrophages. Circulation 2018
Laguna-Fernandez A, Checa A, Carracedo M, …, Thul S, Wheelock CE, Paulsson-Berne G, Ketelhuth DFJ, Hansson GK, Bäck M
(See online at https://doi.org/10.1161/CIRCULATIONAHA.117.032801)
