S100/RAGE-Mediated Inflammation and Modified Cholesterol Lipoproteins as Mediators of Osteoblastic Differentiation of Vascular Smooth Muscle Cells

Abstract

Arterial calcification is a feature of atherosclerosis and shares many risk factors including diabetes, dyslipidemia, chronic kidney disease, hypertension, and age. Although there is overlap in risk factors, anti-atherosclerotic therapies, including statins, fail to reduce arterial, and aortic valve calcifications. This suggests that low density lipoprotein (LDL) may not be the main driver for aortic valve disease and arterial calcification. This review focuses on modified LDLs and their role in mediating foam cell formation in smooth muscle cells (SMCs), with special emphasis on enzyme modified non-oxidized LDL (ELDL). In vivo, ELDL represents one of the many forms of modified LDLs present in the atherosclerotic vessel. Phenotypic changes of macrophages and SMCs brought about by the uptake of modified LDLs overlap significantly in an atherosclerotic milieu, making it practically impossible to differentiate between the effects from oxidized LDL, ELDL, and other LDL modification. By studying in vitro-generated modifications of LDL, we were able to demonstrate marked differences in the transcriptome of human coronary artery SMCs (HCASMCs) upon uptake of ELDL, OxLDL, and native LDL, indicating that specific modifications of LDL in atherosclerotic plaques may determine the biology and functional consequences in vasculature. Enzyme-modified non-oxidized LDL (ELDL) induces calcification of SMCs and this is associated with reduced mRNA levels for genes protective for calcification (ENPP1, MGP) and upregulation of osteoblastic genes. A second focus of this review is on the synergy between hyperlipidemia and accelerated calcification In vivo in a mouse models with transgenic expression of human S100A12. We summarize mechanisms of S100A12/RAGE mediated vascular inflammation promoting vascular and valve calcification in vivo.

Keywords: RAGE (receptor for AGE); S100; calcification; modified LDL; smooth muscle.

Figure 1

Calcified vessels in a female mummy, estimated age 40–45 years old, a princess living in the Seventeenth Dynasty (1580–1550 BCE) of the Second Intermediate Period. Extensive calcification of the aortic and coronary vessels was detected using CT scanning. Reproduced with permission from Elsevier from the Horus Study published in JACC Cardiovascular Imaging (8).

Figure 2

The amount of aortic vascular calcification visualized by Alizarin Red Staining in transgenic mice with S100A12 expression targeted to smooth muscle is modulated by environmental factors. (A) Scant medial calcification in the aorta of 10-month old S100A12/C57Bl6J mice with insert on the lower left for Alizarin Red stain and insert on the lower right with immunofluorescent microscopy staining alpha smooth muscle actin in green. *marks calcification of cartilage of the trachea. Not pictured are wild type C57Bl6 littermate controls, which had no medial calcification; Reproduced with permission from Lippincott Williams and Wilkins (42). (B) Large medial calcification in the aorta of S100A12/C57Bl6J mice with chronic uremia induced by ureter ligation and with insert on the upper right for Alizarin Red stain and insert lower right with Verhoeff van Gieson staining elastic fibers in black. Not pictured are wild type C57BL6J littermate controls with chronic uremia which had no medical calcification; Reproduced with permission from Karger Publishers (43). (C) Advanced atherosclerosis and calcification of atherosclerotic plaques in S100A12 transgenic/ApoE null mice. Not pictured are WT/ApoE littermate controls which had reduced atherosclerosis and calcification; Reproduced with permission from Lippincott Williams and Wilkins (44).

Figure 3

Enzyme modified LDL (ELDL), but not acetylated (acLDL) or oxidized LDL (oxLDL) induces foam cell formation in murine SMCs, in contrast to macrophages which readily take up acLDL, oxLDL and ELDL. In, (A) Oil Red O staining for peritoneal macrophages (upper) and for aortic SMCs (lower) stimulated with 10 μg/ml modified LDL as indicated. (B) Quantification of intracellular cholesterol. (C) Oil Red O staining of aortic SMC stimulated with different concentration of LDLs as indicated and quantified in (D). Reproduced with permission from Lippincott Williams and Wilkins (61). **p < 0.01.

Figure 4

Enzyme modified LDL (ELDL) upregulates phosphate induced calcification in cultured human coronary artery smooth muscle cells. (A) HCASMC cultured in phosphate containing medium (as indicated) with either control BSA or ELDL as indicated. Calcium phosphate deposits were stained with Alizarin Red. (B) Quantification of Ca² deposits. (C) mRNA levels of selected osteoblastic genes in HCASMC incubated for 24 h with either 10 μg/mL ELDL or control BSA with 1 mM inorganic phosphate. Reproduced with permission from Macmillian Publishers Ltd. (62). *p < 0.05; **p < 0.01.

Figure 5

Schematic overview of phenotypic switch of contractile SMCs to osteoblast-like SMC by uptake of enzyme modified LDL in the medial layer and neo-intima in blood vessels. 1a: entry of circulating monocytes and transformation to macrophage foam cells provides enzymatic and inflammatory input for various modifications of native low density lipoprotein (LDL). 1b: Modification of LDL to oxidized LDL (oxLDL) by Phospholipase A2 (sPLA2), Lipoxygenase or Myeloperoxidase. oxLDL is readily taken by macrophages. 2a: Cholesterol esterase together with proteases (plasmin, metalloproteinases (MMPs) or cathepsin) generate non-oxidized enzymatic modified LDL (ELDL). ELDL is taken up by smooth muscle cells via macropinocytosis. 2b: uptake of ELDL activates cell signaling pathways (MAPK, ERK, NFkB) and downregulates mRNA for ectonucleotide pyrophosphatase/phosphodiesterase (ENPP-1), matrix gla protein (MGP) and upregulates mRNA for bone morphogenic protein 2 (BMP2), dentin matrix protein 1 (DMP1), osteopontin (SPP1), and osterix (SP7).

Figure 6

(Upper) Schematic representation of extracellular pyrophosphate metabolisms. Pyrophosphate (PPi) is an important inhibitor of Hydroxyapatide crystal formation. Ectonucleotide pyrophosphate phosphodiesterase 1 (ENPP1) hydrolyzes adenosine triphosphate (ATP) to PPi. PPi is degraded to phosphate (Pi) by tissue nonspecific alkaline phosphatase (TNAP). (Lower) Enzyme modified LDL (ELDL) reduces ENPP1 mRNA and other inhibitors (matrix gla protein, MGP) and increases bone morphogenetic protein 2 and thereby promotes calcification of cultured human coronary artery smooth muscle cells.