TXNIP Suppresses the Osteochondrogenic Switch of Vascular Smooth Muscle Cells in Atherosclerosis

Background: The osteochondrogenic switch of vascular smooth muscle cells (VSMCs) is a pivotal cellular process in atherosclerotic calcification. However, the exact molecular mechanism of the osteochondrogenic transition of VSMCs remains to be elucidated. Here, we explore the regulatory role of TXNIP (thioredoxin-interacting protein) in the phenotypical transitioning of VSMCs toward osteochondrogenic cells responsible for atherosclerotic calcification. Methods: The atherosclerotic phenotypes of Txnip(-/-) mice were analyzed in combination with single-cell RNA-sequencing. The atherosclerotic phenotypes of Tagln-Cre; Txnip(flox/flox) mice (smooth muscle cell-specific Txnip ablation model), and the mice transplanted with the bone marrow of Txnip(-/-) mice were analyzed. Public single-cell RNA-sequencing dataset (GSE159677) was reanalyzed to define the gene expression of TXNIP in human calcified atherosclerotic plaques. The effect of TXNIP suppression on the osteochondrogenic phenotypic changes in primary aortic VSMCs was analyzed. Results: Atherosclerotic lesions of Txnip(-/-) mice presented significantly increased calcification and deposition of collagen content. Subsequent single-cell RNA-sequencing analysis identified the modulated VSMC and osteochondrogenic clusters, which were VSMC-derived populations. The osteochondrogenic cluster was markedly expanded in Txnip(-/-) mice. The pathway analysis of the VSMC-derived cells revealed enrichment of bone- and cartilage-formation-related pathways and bone morphogenetic protein signaling in Txnip(-/-) mice. Reanalyzing public single-cell RNA-sequencing dataset revealed that TXNIP was downregulated in the modulated VSMC and osteochondrogenic clusters of human calcified atherosclerotic lesions. Tagln-Cre; Txnip(flox/flox) mice recapitulated the calcification and collagen-rich atherosclerotic phenotypes of Txnip(-/-) mice, whereas the hematopoietic deficiency of TXNIP did not affect the lesion phenotype. Suppression of TXNIP in cultured VSMCs accelerates osteodifferentiation and upregulates bone morphogenetic protein signaling. Treatment with the bone morphogenetic protein signaling inhibitor K02288 abrogated the effect of TXNIP suppression on osteodifferentiation. Conclusions: Our results suggest that TXNIP is a novel regulator of atherosclerotic calcification by suppressing bone morphogenetic protein signaling to inhibit the transition of VSMCs toward an osteochondrogenic phenotype.

Automated summary

This study reveals that TXNIP plays a regulatory role in the osteochondrogenic transition of vascular smooth muscle cells (VSMCs) and affects the occurrence of atherosclerotic calcification. The effects of TXNIP were analyzed through the phenotypes of Txnip(-/-) mice, Tagln-Cre; Txnip(flox/flox) mice, and mice transplanted with the bone marrow of Txnip(-/-) mice. The downregulation of TXNIP in human calcified atherosclerotic plaques was also confirmed through reanalysis of a public single-cell RNA-sequencing dataset. In vitro experiments showed that suppression of TXNIP promotes osteodifferentiation of VSMCs. These findings suggest that TXNIP regulates atherosclerotic calcification by suppressing bone morphogenetic protein signaling and inhibiting the transition of VSMCs toward an osteochondrogenic phenotype.