ALP Inhibitors Inhibit Inflammatory Responses and Osteoblast Differentiation in hVIC via AKT-ERK Pathways

The aim of this study was to explore the calcification process of aortic valve interstitial cells and its potential association with osteogenic differentiation and alkaline phosphatase (ALP) activity.

The study patients were divided into 3 groups: the control group, the osteogenic induction medium (OM) group and the OM+ALP inhibitor group. Cell calcification was measured by alizarin red S staining and alizarin red S dye released by extracellular matrix (ECM) was quantified by spectrophotometry. Immunohistochemical staining was performed on valve tissues of patients harboring calcified and non-calcified aortic valve disease. Expression of bone morphogenetic protein (BMP), runt related transcription factor 2 (RUNX2), osteocalcin and osteopontin (OPN), was evaluated using immunohistochemistry¸and expression of osteogenic specific markers (BMP, RUNX2 and OPN) was detected using Wesern blot analysis. RNA sequencing was analyzed to further study the exact mechanism of ALP inhibitors in terms of inhibiting the osteogenic differentiation of valvular interstitial cells (VIC). The mRNA levels of tumor necrosis factor alpha (TNF-α), Toll-like receptor 4 (TLR4) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3), were detected using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). In addition, Western blot analysis was performed to evaluate the expression of phosphorylated extracellular regulated protein kinases (ERK), nuclear factor κ B inhibitor α (IκBα) and protein kinase B (AKT) in protein.

Alizarin red staining was positive in the OM and OM+ALP inhibitor groups, and calcified nodules were formed in VIC, which showed a significant difference compared with the control group (P < .05). The semi-quantitative level of calcification in the OM group was higher than in the control group (P < .05), and the semi-quantitative level of calcification in the OM+ALP inhibitor group was lower than in the OM group (P < .05). ALP staining intensity, ALP activity and messenger RNA (mRNA) levels of BMP, RUNX2, osteocalcin, OPN, ERK, IκBα, AKT, TNF-α, Toll-like receptor 4 (TLR4) and NLRP3 inflammasome (NLRP3) in the OM group were higher than in the control group (P < .05). ALP staining intensity, ALP activity and mRNA expressions of BMP, RUNX2, osteocalcin, OPN, phosphorylated ERK, IκBα, AKT, TNF-α and NLRP3 in the OM+ALP inhibitor group were lower than in the OM group (P < .05). Compared with the control group, 723 genes were upregulated and 248 genes were downregulated in the OM group. Compared with the OM group, 352 genes were upregulated and 586 genes were downregulated in the OM+ALP inhibitor group.

We suggest that ALP inhibitors have potential in terms of inhibiting the inflammatory response and osteoblast differentiation of human VIC (hVIC) via the TLR4, AKT, ERK and NLRP3 pathways.