Sources of cells that contribute to atherosclerotic intimal calcification: an in vivo genetic fate mapping study

Abstract

Aims: Vascular cartilaginous metaplasia and calcification are common in patients with atherosclerosis. However, sources of cells contributing to the development of this complication are currently unknown. In this study, we ascertained the origin of cells that give rise to cartilaginous and bony elements in atherosclerotic vessels.

Methods and results: We utilized genetic fate mapping strategies to trace cells of smooth muscle (SM) origin via SM22α-Cre recombinase and Rosa26-LacZ Cre reporter alleles. In animals expressing both transgenes, co-existence within a single cell of β-galactosidase [marking cells originally derived from SM cells (SMCs)] with osteochondrogenic (Runx2/Cbfa1) or chondrocytic (Sox9, type II collagen) markers, along with simultaneous loss of SM lineage proteins, provides a strong evidence supporting reprogramming of SMCs towards osteochondrogenic or chondrocytic differentiation. Using this technique, we found that vascular SMCs accounted for ~80% of Runx2/Cbfa1-positive cells and almost all of type II collagen-positive cells (~98%) in atherosclerotic vessels of LDLr-/- and ApoE-/- mice. We also assessed contribution from bone marrow (BM)-derived cells via analysing vessels dissected from chimerical ApoE-/- mice transplanted with green fluorescence protein-expressing BM. Marrow-derived cells were found to account for ~20% of Runx2/Cbfa1-positive cells in calcified atherosclerotic vessels of ApoE-/- mice.

Conclusion: Our results are the first to definitively identify cell sources attributable to atherosclerotic intimal calcification. SMCs were found to be a major contributor that reprogrammed its lineage towards osteochondrogenesis. Marrow-derived cells from the circulation also contributed significantly to the early osteochondrogenic differentiation in atherosclerotic vessels.

Figure 1

Development of cartilaginous metaplasia and calcification in atherosclerotic vessels of LDLr−/− mice. Aortic arches were dissected from SM22α-Cre+/0:R26R+/0:LDLr−/− mice fed with HFD for 18–20 weeks (A–C) and 24 weeks (D–F). Cells of SM origin were stained by X-gal before embedding. Calcification was stained by the von Kossa method (C and F). Cells of chondrocyte morphology were visualized by Movat pentachrome staining (A, B, D, and E). White arrows designate elastic lamina breakage. White arrowheads designate chondrocyte-like cells. Black arrows designate medial calcification. Asterisk designates necrotic core. L, lumen; I, intima; M, media; Ad, adventitia.

Figure 2

SMCs gave rise to osteochondrogenic precursor- and chondrocyte-like cells in atherosclerotic LDLr−/− vessels. Aortic arches were dissected from SM22α-Cre+/0:R26R+/0:LDLr−/− mice fed with HFD for 20 weeks (A–D) and 28 weeks (E–H). Cells of SMC origin were stained by X-gal before embedding. Adjacent sections were stained by Movat pentachrome (A), von Kossa (E), and immunohistochemistry for SM22α (B and F), Runx2/Cbfa1 (C), Sox9 (G), and Col II (D and H). Insert in G. Higher-powered magnification of the boxed region shows colocalization of β-galactosidase (blue) and chondrocytic transcription factor, Sox9 (brown). L, lumen; I, intima; M, media; Ad, adventitia.

Figure 3

Determination of macrophages in atherosclerotic vessels of LDLr−/− mice. Aortic arches were dissected from SM22α-Cre+/0:R26R+/0:LDLr−/− mice fed with HFD diet for 20 weeks. Cells of SM origin were stained by X-gal before embedding. MOMA-2 antibody was used to identify macrophages (A and B, brown; D and F, green fluorescence). Cells of chondrocyte morphology were visualized by Movat pentachrome staining (C, yellow) and immunohistochemistry for Sox9 (E and F, red fluorescence). Note that MOMA-2-positive cells were not stained blue by X-gal (A and B) and were negative for chondrocyte marker, Sox9 (F). A and B. Images taken from MOMA-2-stained adjacent sections in the boxed regions of C. L, lumen; I, intima; M, media; Ad, adventitia.

Figure 4

Quantitative analysis of osteochondrogenic precursor-like cells in atherosclerotic vessels of LDLr−/− mice. (A). The percentage of Runx2/Cbfa1-positive cells in calcified atherosclerotic vessels of LDLr−/− mice. (B). The percentage of Runx2/Cbfa1-positive cells with β-galactosidase activity. (C–E) Aortic arches of SM22α-Cre+/0:R26R+/0:LDLr−/− mice fed with HFD were stained by X-gal before embedding. Osteochondrogenic precursor cells were stained by Runx2/Cbfa1 antibody. L, lumen; I, intima; M, media; Ad, adventitia.

Figure 5

SMCs gave rise to osteochondrogenic precursor- and chondrocyte-like cells in atherosclerotic ApoE−/− vessels. Aortic arches were dissected from 45-week-old (A–C) and 60-week-old (E–H) SM22α-Cre+/0:R26R+/0:ApoE−/− mice. Cells of SMC origin were stained by X-gal before embedding. Adjacent sections were stained by Movat pentachrome (A), von Kossa (E), and immunohistochemistry for SM22α (B and F), Runx2/Cbfa1 (C), Sox9 (G), and Col II (H). Insert in C and G. Higher-powered magnification of the boxed region shows colocalization of β-galactosidase (blue) with osteochondrogenic marker Runx2/Cbfa1 (C, brown) or with chondrocytic transcription factor, Sox9 (G, brown). (D). The percentage of Runx2/Cbfa1-positive cells in calcified atherosclerotic vessels of LDLr−/− mice. L, lumen; I, intima; M, media; Ad, adventitia.

Figure 6

Transplantation of GFP-positive marrow cells to ApoE−/− mice. GFP marrow cells were transplanted to 35-week-old ApoE−/− mice. Aortic arches were collected for the study 10 weeks after transplantation. Adjacent sections were stained immunohistochemically for GFP (A–C, brown) and Runx2/Cbfa1 (E and F, brown). (D). Quantitative analysis of GFP-positive osteochondrogenic precursor-like cells in atherosclerotic ApoE−/− vessels. Inset in C and F. Higher-powered magnification of the boxed region shows colocalization of GFP (brown, arrows) with osteochondrogenic marker Runx2/Cbfa1 (brown, arrows). L, lumen; I, intima; M, media; Ad, adventitia.