Two-faced Janus: the dual role of macrophages in atherosclerotic calcification.

Calcification is an independent predictor of atherosclerosis-related cardiovascular events. Microcalcification is linked to inflamed, unstable lesions, in comparison to the fibrotic stable plaque phenotype generally associated with advanced calcification. This paradox relates to recognition that calcification presents in a wide spectrum of manifestations that differentially impact plaque's fate. Macrophages, the main inflammatory cells in atherosclerotic plaque, have a multifaceted role in disease progression. They crucially control the mineralization process, from microcalcification to the osteoid metaplasia of bone-like tissue. It is a bilateral interaction that weighs heavily on the overall plaque fate but remains rather unexplored. This review highlights current knowledge about macrophage phenotypic changes in relation to and interaction with the calcifying environment. On the one hand, macrophage-led inflammation kickstarts microcalcification through a multitude of interlinked mechanisms, which in turn stimulates phenotypic changes in vascular cell types to drive microcalcification. Macrophages may also modulate the expression/activity of calcification inhibitors and inducers, or eliminate hydroxyapatite nucleation points. Contrarily, direct exposure of macrophages to an early calcifying milieu impacts macrophage phenotype, with repercussions for plaque progression and/or stability. Macrophages surrounding macrocalcification deposits show a more reparative phenotype, modulating extracellular matrix, and expressing osteoclast genes. This phenotypic shift favours gradual displacement of the pro-inflammatory hubs; the lipid necrotic core, by macrocalcification. Parallels to bone metabolism may explain many of these changes to macrophage phenotype, with advanced calcification able to show homeostatic osteoid metaplasia. As the targeted treatment of vascular calcification developing in atherosclerosis is thus far severely lacking, it is crucial to better understand its mechanisms of development.

Proteoglycan 4 Modulates Osteogenic Smooth Muscle Cell Differentiation during Vascular Remodeling and Intimal Calcification.

Calcification is a prominent feature of late-stage atherosclerosis, but the mechanisms driving this process are unclear. Using a biobank of carotid endarterectomies, we recently showed that Proteoglycan 4 (PRG4) is a key molecular signature of calcified plaques, expressed in smooth muscle cell (SMC) rich regions. Here, we aimed to unravel the PRG4 role in vascular remodeling and intimal calcification. PRG4 expression in human carotid endarterectomies correlated with calcification assessed by preoperative computed tomographies. PRG4 localized to SMCs in early intimal thickening, while in advanced lesions it was found in the extracellular matrix, surrounding macro-calcifications. In experimental models, Prg4 was upregulated in SMCs from partially ligated ApoE-/- mice and rat carotid intimal hyperplasia, correlating with osteogenic markers and TGFb1. Furthermore, PRG4 was enriched in cells positive for chondrogenic marker SOX9 and around plaque calcifications in ApoE-/- mice on warfarin. In vitro, PRG4 was induced in SMCs by IFNg, TGFb1 and calcifying medium, while SMC markers were repressed under calcifying conditions. Silencing experiments showed that PRG4 expression was driven by transcription factors SMAD3 and SOX9. Functionally, the addition of recombinant human PRG4 increased ectopic SMC calcification, while arresting cell migration and proliferation. Mechanistically, it suppressed endogenous PRG4, SMAD3 and SOX9, and restored SMC markers' expression. PRG4 modulates SMC function and osteogenic phenotype during intimal remodeling and macro-calcification in response to TGFb1 signaling, SMAD3 and SOX9 activation. The effects of PRG4 on SMC phenotype and calcification suggest its role in atherosclerotic plaque stability, warranting further investigations.