Calcific aortic valve disease (CAVD) is the most prevalent heart valve disorder in the elderly. Valvular fibrocalcification is a characteristic pathological change. In diseased valves, monocyte accumulation is evident, and aortic valve interstitial cells (AVICs) display greater fibrogenic and osteogenic activities. However, the impact of activated monocytes on valular fibrocalcification remains unclear. We tested the hypothesis that pro-inflammatory mediators from activated monocytes elevate AVIC fibrogenic and osteogenic activities.
Picro-sirius red staining and Alizarin red staining revealed collagen and calcium depositions in cultured human AVICs exposed to conditioned media derived from Pam3CSK4-stimulated monocytes (Pam3 CM). Pam3 CM up-regulated alkaline phosphatase (ALP), an osteogenic biomarker, and extracellular matrix proteins collagen I and matrix metalloproteinase-2 (MMP-2). ELISA analysis identified high levels of RANTES and TNF-α in Pam3 CM. Neutralizing RANTES in the Pam3 CM reduced its effect on collagen I and MMP-2 production in AVICs while neutralizing TNF-α attenuated the effect on AVIC ALP production. In addition, Pam3 CM induced NF-κB and JNK activation. While JNK mediated the effect of Pam3 CM on collagen I and MMP-2 production, NF-κB was critical for the effect of Pam3 CM on ALP production in AVICs.
This study demonstrates that activated monocytes elevate the fibrogenic and osteogenic activities in human AVICs through a paracrine mechanism. TNF-α and RANTES mediate the pro-fibrogenic effect of activated monocytes on AVICs through activation of JNK, and TNF-α also activates NF-κB to elevate AVIC osteogenic activity. The results suggest that infiltrated monocytes elevate AVIC fibrocalcific activity to promote CAVD progression.
© 2022. The Author(s).

Calcific aortic valve disease (CAVD) is an age-related and slowly progressive valvular disorder. Overexpression of matrix metalloproteinase 12 (MMP-12) has been found in atherosclerosis, stiffed vascular tissue, and calcified aortic valves. We hypothesized that MMP-12 may induce the pro-osteogenic responses in human aortic valve interstitial cells (AVICs).
Human AVICs were isolated from normal and calcified aortic valves. Cells were treated with MMP-12. The pro-osteogenic marker Runt-related transcription factor 2 (RUNX-2), bone morphogenetic protein 2 (BMP-2), and alkaline phosphatase (ALP), as well as MMP-12-associated signaling molecules, were analyzed.
Human calcified aortic valves expressed significantly higher MMP-12 than normal human aortic valves. MMP-12-induced the expression of RUNX-2, BMP-2, ALP, and calcium deposit formation. Suppression of MMP-12 by its inhibitor decreased the expression of RUNX-2, BMP-2, and ALP. MMP-12-induced osteogenic responses were associated with higher levels of phosphorylation of p38 mitogen-activated protein kinases (MAPK), low density lipoprotein-related protein 6 (LRP-6), and β-catenin signaling molecules. Calcified aortic valves exhibited markedly higher levels of LRP-6 and β-catenin levels. Inhibition of either p38 MAPK or LRP-6 attenuated MMP-12-induced expression of RUNX-2, BMP-2, and ALP. Suppression of p38 MAPK abrogated MMP-12-induced activation of LRP-6 and β-catenin signaling pathways.
MMP-12 induces pro-osteogenic responses in AVICs by activation of p38 MAPK-mediated LRP-6 and β-catenin signaling pathways. The study revealed that the potential role of MMP-12 in the pathogenesis of CAVD and therapeutically targeting MMP-12 may suppress the development of CAVD.
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