Osteoclast-Like Cells in Aneurysmal Disease Exhibit an Enhanced Proteolytic Phenotype

Abstract

Abdominal aortic aneurysm (AAA) is among the top 20 causes of death in the United States. Surgical repair is the gold standard for AAA treatment, therefore, there is a need for non-invasive therapeutic interventions. Aneurysms are more closely associated with the osteoclast-like catabolic degradation of the artery, rather than the osteoblast-like anabolic processes of arterial calcification. We have reported the presence of osteoclast-like cells (OLCs) in human and mouse aneurysmal tissues. The aim of this study was to examine OLCs from aneurysmal tissues as a source of degenerative proteases. Aneurysmal and control tissues from humans, and from the mouse CaPO₄ and angiotensin II (AngII) disease models, were analyzed via flow cytometry and immunofluorescence for the expression of osteoclast markers. We found higher expression of the osteoclast markers tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP-9), and cathepsin K, and the signaling molecule, hypoxia-inducible factor-1α (HIF-1α), in aneurysmal tissue compared to controls. Aneurysmal tissues also contained more OLCs than controls. Additionally, more OLCs from aneurysms express HIF-1α, and produce more MMP-9 and cathepsin K, than myeloid cells from the same tissue. These data indicate that OLCs are a significant source of proteases known to be involved in aortic degradation, in which the HIF-1α signaling pathway may play an important role. Our findings suggest that OLCs may be an attractive target for non-surgical suppression of aneurysm formation due to their expression of degradative proteases.

Keywords: aneurysm; hypoxia-inducible factor-1α (HIF-1α); osteoclast-like cell (OLC).

Figure 1

Flow cytometry gating strategy. Cells derived from culture or tissue samples were gated for size, singlets, and viability. Live cells were then analyzed for their expression of CD11b, TRAP, cathepsin K, MMP-9, and HIF-1α. The CD11b⁺ population was divided into TRAP⁻ and TRAP⁺ populations which were analyzed for their expression of cathepsin K and MMP-9.

Figure 2

Treatment of macrophages with CaPO₄ and TNFα results in osteoclastogenesis and increased protease production. RAW 264.7 macrophages were treated with CaPO₄ and TNFα and analyzed for the percent of live cells expressing TRAP (A), cathepsin K (B), MMP-9 (C), and HIF-1α (D). (E,F) The expression (MFI) of cathepsin K and MMP-9, respectively, in TRAP⁺ cells (TPM) compared to TRAP⁻ cells (macrophage). The data represent three independent experiments. Data are expressed as mean ± SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001. MFI, median fluorescence intensity.

Figure 3

Mouse CaPO₄ and AngII-induced aneurysms. (A,B) Representative images of mice treated via perivascular application of CaPO₄ or subcutaneous administration of AngII, respectively. Black arrows indicate aneurysmal vessels. (C,D) CaPO₄ and AngII treatment resulted in significant vessel dilation compared to control vessels (n = 5). In the CaPO₄ model (C), the contralateral carotid arteries served as controls (n = 5), and PBS-only treated mice served as controls for the AngII model (n = 3) (D). These data are expressed as mean ± SEM. * p < 0.05, **** p < 0.0001.

Figure 4

Cells from mouse CaPO₄ (A), mouse AngII (B), and human (C) aneurysms exhibit increased expression of the myeloid marker, CD11b, and the osteoclastogenic markers TRAP, cathepsin K, and MMP-9. Tissues from CaPO₄ mice (n = 5), AngII mice (n = 6) and PBS-only controls (n = 3), human AAA (n = 4), and human carotid plaques (n = 3) were enzymatically digested and processed to obtain single cell suspensions for flow cytometric analysis, as described in the Methods section. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. AAA, abdominal aortic aneurysm.

Figure 5

Increased expression of HIF-1α in mouse CaPO₄ (A,D), mouse AngII (B,E), and human (C,F) aneurysms. Tissues from CaPO₄ mice (n = 5), AngII mice (n = 5) and PBS-only controls (n = 3), human AAA (n = 4), and human carotid plaques (n = 3) were enzymatically digested and processed to obtain single cell suspensions for flow cytometric analysis, as described in the Methods section. (A–C) depict the percentage of live cells expressing HIF-1α in aneurysmal and control tissues. (D–F) compare the percentage of live cells expressing HIF-1α between myeloid cells (CD11b⁺, TRAP⁻) and TPMs (CD11b⁺, TRAP⁺) from aneurysmal tissues. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. TPM, TRAP-positive macrophage. AAA, abdominal aortic aneurysm.

Figure 6

Mouse and human aneurysms contain more TPMs than non-aneurysmal control tissues. Mouse CaPO₄ (A), mouse AngII (B), and human (C) aneurysms exhibit more TPMs (cells double-positive for expression of the myeloid marker, CD11b, and the osteoclast marker, TRAP) than respective controls. (A–C) show TPMs as a percentage of live cells in tissues from CaPO₄ mice (n = 5) (A), AngII mice (n = 6) and PBS-only controls (n = 3) (B), human AAA (n = 4) and human carotid plaques (n = 3) (C). Tissues were enzymatically digested and processed to obtain single-cell suspensions for flow cytometric analysis, as described in the Methods section. (D–I) Immunofluorescence staining of frozen control and aneurysmal sections. Control sections (D–F), and aneurysmal sections (G–I) were stained for CD11b (red), TRAP (green), and DAPI (blue) as described in the Methods section. Yellow arrows indicate TPMs. 100× magnification (E,H), 200× magnification (D,F,I), 400× magnification (G). Data are expressed as mean ± SEM. ** p < 0.01, *** p < 0.001. TPM, TRAP-positive macrophage. AAA, abdominal aortic aneurysm.

Figure 7

TRAP-positive macrophages produce more proteases than myeloid cells in mouse CaPO₄ (A,D), mouse AngII (B,E), and human (C,F) aneurysms. Aneurysmal tissues from CaPO₄-treated mice (n = 4), AngII-treated mice (n = 6), and human AAA (n = 4) were enzymatically digested and processed to obtain single-cell suspensions for flow cytometric analysis, as described in the Methods section. The expression of cathepsin K (A–C) and MMP-9 (D–F) is compared between myeloid cells (CD11b⁺, TRAP⁻) and TPMs (CD11b⁺, TRAP⁺). Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. TPM, TRAP-positive macrophage. AAA, abdominal aortic aneurysm.