Cellular FXIII in Human Macrophage-Derived Foam Cells

Abstract

Macrophages express the A subunit of coagulation factor XIII (FXIII-A), a transglutaminase which cross-links proteins through Nε-(γ-L-glutamyl)-L-lysyl iso-peptide bonds. Macrophages are major cellular constituents of the atherosclerotic plaque; they may stabilize the plaque by cross-linking structural proteins and they may become transformed into foam cells by accumulating oxidized LDL (oxLDL). The combination of oxLDL staining by Oil Red O and immunofluorescent staining for FXIII-A demonstrated that FXIII-A is retained during the transformation of cultured human macrophages into foam cells. ELISA and Western blotting techniques revealed that the transformation of macrophages into foam cells elevated the intracellular FXIII-A content. This phenomenon seems specific for macrophage-derived foam cells; the transformation of vascular smooth muscle cells into foam cells fails to induce a similar effect. FXIII-A containing macrophages are abundant in the atherosclerotic plaque and FXIII-A is also present in the extracellular compartment. The protein cross-linking activity of FXIII-A in the plaque was demonstrated using an antibody labeling the iso-peptide bonds. Cells showing combined staining for FXIII-A and oxLDL in tissue sections demonstrated that FXIII-A-containing macrophages within the atherosclerotic plaque are also transformed into foam cells. Such cells may contribute to the formation of lipid core and the plaque structurization.

Keywords: atherosclerotic plaque; cross-linking; enzyme-modified LDL; factor XIII; foam cells; macrophages; oxidized LDL; transglutaminase; vascular smooth muscle cells.

Figure 1

Investigation of FXIII-A expression in foam cells of different origin. (A) FXIII-A in macrophages transformed into foam cells by oxidized LDL ingestion. FXIII-A appears in green, while oxidized LDL is shown in red color. Scale bars correspond to 10 μm. (B) The lack of FXIII-A in vascular smooth muscle cells transformed into foam cells by ingestion of enzyme-modified non-oxidized LDL. Smooth muscle actin is depicted in green, while modified LDL is represented by red color. Scale bars correspond to 50 μm.

Figure 2

Elevation of FXIII-A in macrophages following their transformation into foam cells by a single dose of 50 μg/mL oxidized LDL (oxLDL). (A) FXIII-A was measured by ELISA in the lysates of non-treated cells and in cells harvested 24, 48, and 72 h after the addition of oxLDL. Statistical significance was calculated by comparing the results of oxLDL-treated cells to those of non-treated cells (n = 5, * p < 0.05). (B) A representative Western blot demonstrates an elevated FXIII-A level in macrophages transformed into foam cells. The arrows indicate the migration of 75 kDa and 100 kDa components of the molecular weight markers in the Precision Plus Protein Standards.

Figure 3

Macrophages in carotid artery plaque. (A) Type IV carotid artery plaque, with clearly visible lipid core, most of which is within the elliptical border. The lipid core is surrounded by CD68-positive macrophages. (B) FXIII-A-positive cells are detected in the plaque by immune-peroxidase staining; FXIII-A is also present extracellularly in the lipid core. More details are shown in Figure 4.

Figure 4

Immunohistochemical visualization of FXIII-A and cross-linked protein structures in the atherosclerotic plaque. (A) FXIII-A of cellular and extracellular localization in the atherosclerotic plaque. (B) FXIII-A-containing macrophages in the plaque at higher magnification. (C) Negative control for FXIII-A staining. (D) Protein mass cross-linked through Nε-(γ-L-glutamyl)-L-lysyl bonds in a non-cellular part of the sclerotic plaque.

Figure 5

Atheromatous plaque cryosections with combined Oil Red O (ORO) and FXIII-A staining (PolyDetector HRP Green chromogen). (A) Numerous macrophages, some with foamy cytoplasm, in which the presence of ORO-positive vacuoles can be observed. ORO-positive droplets are also present in the extracellular compartment. (B) Examples of two FXIII-A-positive macrophages which were also stained with ORO.