Neointima formed by arterial smooth muscle cells expressing versican variant V3 is resistant to lipid and macrophage accumulation

Abstract

Objective: Extracellular matrix (ECM) of neointima formed following angioplasty contains elevated levels of versican, loosely arranged collagen, and fragmented deposits of elastin, features associated with lipid and macrophage accumulation. ECM with a low versican content, compact structure, and increased elastic fiber content can be achieved by expression of versican variant V3, which lacks chondroitin sulfate glycosaminoglycans. We hypothesized that V3-expressing arterial smooth muscle cells (ASMC) can be used to form a neointima resistant to lipid and macrophage accumulation associated with hypercholesterolemia.

Methods and results: ASMC transduced with V3 cDNA were seeded into ballooned rabbit carotid arteries, and animals were fed a chow diet for 4 weeks, followed by a cholesterol-enriched diet for 4 weeks, achieving plasma cholesterol levels of 20 to 25 mmol/L. V3 neointimae at 8 weeks were compact, multilayered, and elastin enriched. They were significantly thinner (57%) than control neointimae; contained significantly more elastin (118%), less collagen (22%), and less lipid (76%); and showed significantly reduced macrophage infiltration (85%). Mechanistic studies demonstrated that oxidized low-density lipoprotein stimulated the formation of a monocyte-binding ECM, which was inhibited in the presence of V3 expressing ASMC.

Conclusion: These results demonstrate that expression of V3 in vessel wall creates an elastin-rich neointimal matrix that in the presence of hyperlipidemia is resistant to lipid deposition and macrophage accumulation.

Figure 1. Feeding protocol and plasma cholesterol levels

Dietary protocol, and plasma cholesterol levels (means ± SEM) for vector control and V3 groups over 8-week period following balloon angioplasty and cell seeding.

Figure 2. Neointimal morphology

A, Mean (± SEM) neointimal widths for vector control and V3 vessels. B and C, Orcein-stained (orange/brown) neointimae showing scattered elastin deposits (arrow) in vector control (B) and multiple layers of organized fibers (arrow) in V3 seeded vessels (C). D and E, Elastin autofluorescence (green) showing diffuse neointimal fluorescence (arrow) of scattered elastin deposits in vector control (D) and strongly fluorescent elastic fibers (arrow) in V3 neointima (E). Note autofluorescence of internal elastic lamina (IEL) in E. F and G, Eight-week vector control (F) and V3 (G) neointimae immunostained with 9BA12 for chondroitin sulfate (chromogen DAB) showing reduced CS staining in V3 neointimae compared with control neointimae. Panels counterstained with haematoxylin. Arrows indicate position of the IEL. Scale bars B, D, F and G 20μm; C and E 10μm.

Figure 3. Neointimal ultrastructure

A, B, and C, toluidine blue stained (A) and electron micrographic (B and C) images of vector control 8-week neointima. Subendothelial zone adjacent to lumen (∟) (A and B) contains lipid-rich foam cells (*) and disorganized ECM containing small scattered deposits of elastin (arrow) (B). Mid-intimal zone (A and C) contains increased amount and larger deposits of elastin (dark purple/blue stained deposits in A, arrows in C), in an open matrix containing small collagen fibrils between ASMC. Purple hue in A due to metachromasia by CSPG. Labelled boxes in A and D indicate position of electron micrographs B, C, E, and F. Corresponding images of V3 neointima showing multiple, circumferential bands of elastin throughout full thickness (D) of compact neointima from luminal surface to IEL. Differentiated, elongated, closely packed ASMC in subendothelial (E) and mid-intimal regions (F) are separated by bands of elastin, similar in arrangement to medial lamellae. Open arrows in F indicate bi-lamina elastin structure, shown at higher magnification in G, typical of developing medial elastic lamellae. H, I, and J, Volume fractions (%) for cells and ECM (H), and for ECM components elastin, collagen and matrix (I and J), in 8-week vector control and V3 neointimae determined by point counting of electron micrographs. V3 neointimae contained significantly more elastin, and less collagen and matrix, compared with vector controls (I). Elevated elastin content in V3 neointimae was similar for each of the three intimal zones, in contrast to vector control where the subendothelial zone contained significantly (paired t test) less elastin than mid- and deep-neointimal zones (O). Error bars SEM. Scale bars (A and D) 20 μm, (B and E) 2μm, (C and F) 1μm.

Figure 4. Lipid deposition

A and B, Lipid content (area analysis of Oil Red O staining) (mean ± SEM) of vector control and V3 neointimae, expressed as total lipid per area (A) and as a % of neointimal area (B). C and E, Representative cross-sections of vector control (C) and V3-seeded (E) carotid arteries stained with Oil Red O, showing reduced lipid and thinner neointima in V3-seeded vessel. Scale bars 200μm. D and F, Oil Red O stained neointimae showing lipid deposition throughout neointima of vector control (D) and absence of lipid in V3 neointima (F). Scale bars D 20μm; F 10μm.

Figure 5. Macrophages distribution and content

A, B, and C, Vector control carotid cross sections stained for lipid (Oil Red O) (A), and for macrophages (anti-RAM11) visualised with Nova Red (B and C), showing association between lipid staining and macrophages. Macrophages are concentrated in the subendothelial zone, but are also present in the deep neointima and in the media below the IEL (C). D-H, Corresponding images for V3-seeded carotid, showing absence of lipid (D) and macrophages (E and F). G and H, Small focal deposits of subendothelial macrophages in V3-seeded vessel. Paired images (A and B; D and E) are from closely adjacent tissue sections. Panels C, F, and H are enlargements of boxed areas in B, E, and G, respectively. Panels B, C, and E–H counterstained with haematoxylin. Arrows indicate position of IEL. Scale bars in A, B, D, E, and G 200μm. I, Morphometric analysis of macrophage content, expressed as % area occupied, of total wall (neointima plus media), neointima, and media, in vector control and V3 vessels.

Figure 6. Monocyte adherence in vitro

A Hyaluronidase-sensitive monocyte binding to 21-day cultures of vector control and V3 ASMC in untreated cultures, and cultures stimulated with 5μg/ml of tunicamycin or 5μg/ml of oxLDL for 20 hrs, showing reduced binding to matrix generated by V3-expressing ASMC. B Monocyte binding to polyacrylamide gels treated with collagen and elastin, showing significantly reduced binding to elastin compared with collagen.