Toll-like receptor 9 promotes aortic chondrogenesis and calcification in apolipoprotein E-deficient mice.

Vascular calcification represents a significant clinical challenge, leading to cardiovascular disease, though its underlying mechanisms remain incompletely understood. Recent studies indicate that Toll-like receptor 9 (TLR9), a key element of innate immunity, plays a pathogenic role in vascular inflammation and atherogenesis. Therefore, we hypothesized that TLR9 signaling promotes vascular chondrogenesis and calcification. We compared apolipoprotein E-deficient (ApoE-/-) mice and Tlr9-/- ApoE-/- mice after 24 -weeks high-cholesterol diet feeding. There were no differences between the groups in body weight gain, blood pressure, or plasma glucose levels, although total cholesterol levels were significantly lower in the Tlr9-/- ApoE-/- mice. The genetic deletion of TLR9 attenuated vascular calcification as determined by von Kossa staining (5.83 ± 1.14% vs. 3.04 ± 0.68%; P < 0.05), alkaline phosphatase (ALP-1) activity (P < 0.05), and chondroid matrix deposition as determined by Alcian blue staining (P < 0.05) in aortic arch compared with control mice. Immunohistostaining revealed that TLR9 deletion also decreased bone morphogenetic protein (BMP)-2 expression in aortic plaques (P < 0.05). In vitro experiments revealed that TLR9 activation by ODN1826, a TLR9 agonist, stimulated BMP-2 expression in murine peritoneal macrophages, but not in Tlr9-deficient macrophages. Although TLR9 agonists had no direct effect on vascular smooth muscle cells (VSMCs), the culture supernatants of macrophages stimulated with TLR9 agonist increased BMP-2 expression in VSMCs. TLR9 signaling promotes vascular chondrogenesis and calcification in ApoE-/- mice. Our analyses suggest that TLR9 pathway contributes to bone morphogenic activation of macrophages and VSMCs at least partially, participating in the development of vascular calcification.