The Interplay of Wnt‐ and PPARγ‐signaling in macrophages & vascular smooth muscle cells during vascular calcification

Abstract

BackgroundMolecular mechanisms and structural changes of the vessel wall including inflammation and vascular calcification (VC) are linked to atherosclerosis, and various cell types such as macrophages and vascular smooth muscle cells (VSMC) are involved. Importantly, VC enhances the risk of acute cardiovascular events by 4‐fold. Hence, it is of great importance to reveal underlying molecular mechanisms to prevent this outcome. The environmental shifts in atherosclerotic plaques stimulate the expression of Wnt5a and BMP‐2. Both factors are secreted by M1‐macrophages and induce an osteo‐chondrogenic phenotype of VSMC by enhancing SOX9 and RUNX2 mRNA expression while modulating the Wnt‐signaling pathway. Subsequently, we observed a 2‐fold upregulation of Wnt5a expression in human macrophages treated with serum from patients with acute myocardial infarction. However, PPARγ‐signaling drives the secretion of Wnt‐inhibitors (Dkk1, sFRPs) in multiple cell types. This raises the question if PPARγ‐stimulation can limit the deleterious effects of pathologically activated Wnt‐signaling in VC by attenuating the osteogenic differentiation of VSMC. Thus, identification of the molecular pathways linking PPARγ‐and Wnt‐signaling may be of major clinical importance for the treatment of VC. Additionally, the existence of already registered PPARγ‐agonists could receive new clinical indications.MethodsHuman VSMC from abdominal aorta were differentiated to synthetic VSMC (PDGF‐BB 10 ng/ml, FCS 20%, 3 d) and calcified for 12 d (Pi, 2 mM; Cai, 3 mM, FCS 5%). THP‐1 cells derived M0‐macrophages (PMA, 150 nM, 24 h) were activated to M1‐macrophages (IFN‐γ, 20 ng/ml; LPS 10 ng/ml; 24 h). Cells were treated with a PPARγ‐agonist (Rosiglitazone, 10 μM) or PPARγ‐antagonist (GW9662, 10 μM) for 12 d (VSMC) or 24 h (macrophages).ResultsPPARγ‐stimulation decreased the expression of osteo‐chondrogenic genes (SOX9 −50%; RUNX2 −37%, P<0.01), WNT5A (−31%, P<0.05) and nuclear translocation of active β‐catenin (−20%, P<0.01), but not calcium‐deposition in calcifying, synthetic VSMC. The Wnt/β‐catenin‐Inhibitor DKK1 was increased (+46%, P<0.05) and blocking PPARγ increased calcium‐deposition (+78%, P<0.05). In M1‐macrophages, PPARγ was decreased (−73%, P<0.0001) and WNT5A increased (+424%, P<0.0001) while BMP2 was not affected. However, PPARγ‐stimulation reduced BMP2 mRNA expression (−29%, P<0.01).ConclusionsPPARó‐stimulation decreases mRNA expression of BMP2 in M1‐macrophages, which may prevent secretion while possibly reducing osteogenic differentiation of VSMC. Moreover, PPARγ‐stimulation attenuated Wnt5a‐ and Wnt/β‐catenin‐signaling by modulating DKK1 expression and translocation of active β‐catenin in synthetic, calcifying VSMC. Hence, these effects may attenuate the osteogenic differentiation of VSMC and subsequent calcification of atherosclerotic plaques.Support or Funding InformationThis project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska‐Curie grant agreement No 722609.