Hedgehog signaling is a potential therapeutic target for vascular calcification.

• Vascular calcification (VC) is prevalent in patients with CKD and contributes to their increased risk of all-cause mortality. • DEGs in VC is significantly enriched in the process of ossification, osteoblast differentiation, and Hedgehog signaling pathway. • PPI network analysis identified top 10 key genes involved in the development of VC. • CMAP analysis predicted several small molecular drugs targeting these key genes including SAG and CPN. • Our in vitro experiments confirmed that SAG markedly alleviated VSMC calcification and CPN significantly exacerbated VSMC calcification. Patients with chronic kidney disease (CKD) suffered from vascular calcification (VC), one major contributor for their increased mortality rate. Hedgehog (Hh) signaling plays a crucial role in physiological bone mineralization and is associated with several cardiovascular diseases. However, the molecular changes underlying VC is ill defined and it remains unclear whether Hh signaling intervention affects VC. We constructed human primary vascular smooth muscle cell (VSMC) calcification model and performed RNA sequencing. Alizarin red staining and calcium content assay were conducted to identify the occurrence of VC. Three different R packages were applied to determine differentially expressed genes (DEGs). Enrichment analysis and protein–protein interaction (PPI) network analysis were carried out to explore the biological roles of DEGs. qRT-PCR assay was then applied to validate the expression of key genes. By using Connectivity Map (CMAP) analysis, several small molecular drugs targeting these key genes were obtained, including SAG (Hedgehog signaling activator) and cyclopamine (CPN) (Hedgehog signaling inhibitor), which were subsequently used to treat VSMC. Obvious Alizarin red staining and increased calcium content identified the occurrence of VC. By integrating results from three R packages, we totally obtained 166 DEGs (86 up-regulated and 80 down-regulated), which were significantly enriched in ossification, osteoblast differentiation, and Hh signaling. PPI network analysis identified 10 key genes and CMAP analysis predicted several small molecular drugs targeting these key genes including chlorphenamine, isoeugenol, CPN and phenazopyridine. Notably, our in vitro experiment showed that SAG markedly alleviated VSMC calcification, whereas CPN significantly exacerbated VC. Our research provided deeper insight to the pathogenesis of VC and indicated that targeting Hh signaling pathway may represent a potential and effective therapy for VC. [ABSTRACT FROM AUTHOR]