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 thioredoxin-interacting protein (TXNIP) 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.

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Background Hydroxyapatite (HAp) possesses osteoconductive properties, and its granular form can serve as an effective drug delivery vehicle for bone regeneration. Quercetin (Qct), a plant-derived bioflavonoid, is known to promote bone regeneration; however, its comparative and synergistic effects with the commonly used bone morphogenetic protein-2 (BMP-2) have not been investigated. Methods We examined the characteristics of newly formed HAp microbeads using an electrostatic spraying method and analyzed the in vitro release pattern and osteogenic potential of ceramic granules containing Qct, BMP-2, and both. In addition, HAp microbeads were transplanted into a rat critical-sized calvarial defect and the osteogenic capacity was assessed in vivo. Results The manufactured beads had a microscale size of less than 200 μm, a narrow size distribution, and a rough surface. The alkaline phosphatase (ALP) activity of osteoblast-like cells cultured with the BMP-2-and-Qct-loaded HAp was significantly higher than that of either Qct- or BMP-2-loaded HAp groups. The mRNA levels of osteogenic marker genes such as ALP and runt-related transcription factor 2 were found to be upregulated in the HAp/BMP-2/Qct group compared to the other groups. In micro-computed tomographic analysis, the amount of newly formed bone and bone surface area within the defect was significantly higher in the HAp/BMP-2/Qct group, followed by the HAp/BMP-2 and HAp/Qct groups, which is consistent with the histomorphometrical results. Conclusions These results imply that electrostatic spraying can be an efficient strategy to produce homogenous ceramic granules and that the BMP-2-and-Qct-loaded HAp microbeads can serve as effective implants for bone defect healing.

Hydrocephalus is one of the most common central nervous system malformations in domestic dogs, yet they are poorly documented and studied in wild carnivoran mammals. A pup of raccoon dog (Nyctereutes procyonoides) was rescued and brought to Wildlife Center. The pup showed generalized ataxia, a domed skull, and an open bregmatic fontanelle. Ultrasound and MRI showed severe enlargement of the lateral ventricle with the loss of septum pellucidum resulting in a single large ventricle and cervical syringohydromyelia. Although treatment was attempted, the animal was euthanized due to poor prognosis. At necropsy, macroscopic findings were identical to the diagnostic imaging, where marked enlargement of the calvarium, and attenuated gyri and sulci were observed. Finally, hydrocephalus was confirmed. Here, we describe a case of hydrocephalus in a raccoon dog (Nyctereutes procyonoides).