MicroRNA-223-3p inhibits vascular calcification and the osteogenic switch of vascular smooth muscle cells.

Vascular calcification is the ectopic deposition of calcium hydroxyapatite minerals in arterial wall, which involves the transdifferentiation of vascular smooth muscle cells (VSMCs) toward an osteogenic phenotype. However, the underlying molecular mechanisms regulating the VSMC osteogenic switch remain incompletely understood. In this study, we examined the roles of microRNAs (miRNAs) in vascular calcification. miRNA-seq transcriptome analysis identified miR-223-3p as a candidate miRNA in calcified mouse aortas. MiR-223-3p knockout aggravated calcification in both medial and atherosclerotic vascular calcification models. Further, RNA-seq transcriptome analysis verified JAK-STAT and PPAR signaling pathways were upregulated in both medial and atherosclerotic calcified aortas. Overlapping genes in these signaling pathways with predicted target genes of miR-223-3p derived from miRNA databases, we identified signal transducer and activator of transcription 3 (STAT3) as a potential target gene of miR-223-3p in vascular calcification. In vitro experiments showed that miR-223-3p blocked interleukin-6 (IL-6)/STAT3 signaling, thereby preventing the osteogenic switch and calcification of VSMCs. In contrast, overexpression of STAT3 diminished the effect of miR-223-3p. Taken together, the results indicate a protective role of miR-223-3p that inhibits both medial and atherosclerotic vascular calcification by regulating IL-6/STAT3 signaling-mediated VSMC transdifferentiation.

Age-related decline of interferon-gamma responses in macrophage impairs satellite cell proliferation and regeneration.

BACKGROUND: Impaired muscle regeneration and increased muscle fibrosis are observed in aged muscle accompanied by progressive loss of muscle mass (sarcopenia). However, the underlying mechanism is still unclear. METHODS: The differentiated expressed genes in young and aged muscles after acute injury by cardiotoxin were identified by RNA-sequence analysis. Single-cell RNA-sequence analysis was used to identify cell clusters and functions in young muscle after acute injury, and flow cytometry analysis and sorting were used to validate the function. The proliferation and differentiation functions of satellite cells were accessed by immunostaining with 5-ethynyl-2'-deoxyuridine and embryonic myosin heavy chain (eMyHC), respectively. Muscle regeneration ability was accessed by histopathological and molecular biological methods. RESULTS: Gene expression patterns associated with responses to interferon-gamma (IFN-γ) (15 genes; false discovery rate < 0.001) were significantly down-regulated during muscle regeneration in aged mice (P = 2.25e-7). CD8+ T cells were the main source of increased IFN-γ after injury, adoptive transfer of wild-type CD8+ T cells to IFN-γ-deficient young mice resulted in 78% increase in cross-sectional areas (CSAs) of regenerated myofibres (P < 0.05) and 63% decrease in muscle fibrosis (P < 0.05) after injury. Single-cell RNA-sequence analysis identified a novel subset of macrophages [named as IFN-responsive macrophages (IFNRMs)] that specifically expressed IFN-responsive genes (Ifit3, Isg15, Irf7, etc.) in young mice at 3 days after injury, and the number of this macrophage subset was ~20% lower in aged mice at the same time (P < 0.05). IFNRMs secreted cytokine C-X-C motif chemokine 10 (CXCL10) that promoted the proliferation and differentiation of satellite cells via its receptor, CXCR3. Intramuscular recombinant CXCL10 treatment in aged mice rejuvenated the proliferation of satellite cells (80% increase in Ki-67+ Pax7+ cells, P < 0.01) and resulted in 27% increase in CSA of regenerated myofibres (P < 0.01) and 29% decrease in muscle fibrosis (P < 0.05). CONCLUSIONS: Our study indicates that decline in IFN-γ response in a novel subset of macrophage contributes to satellite cells dysfunctions in aged skeletal muscles and demonstrates that this mechanism can be targeted to restore age-associated myogenesis.

MicroRNA-223-3p promotes skeletal muscle regeneration by regulating inflammation in mice.

After injury, the coordinated balance of pro- and anti-inflammatory factors in the microenvironment contribute to skeletal muscle regeneration. However, the underlying molecular mechanisms regulating this balance remain incompletely understood. In this study, we examined the roles of microRNAs (miRNAs) in inflammation and muscle regeneration. miRNA-Seq transcriptome analysis of mouse skeletal muscle revealed that miR-223-3p is upregulated in the early stage of muscle regeneration after injury. miR-223-3p knockout resulted in increased inflammation, impaired muscle regeneration, and increased interstitial fibrosis. Mechanistically, we found that myeloid-derived miR-223-3p suppresses the target gene interleukin-6 (Il6), associated with the maintenance of the proinflammatory macrophage phenotype during injury. Administration of IL-6-neutralizing antibody in miR-223-3p-knockout muscle could rescue the impaired regeneration ability and reduce the fibrosis. Together, our results reveal that miR-223-3p improves muscle regeneration by regulating inflammation, indicating that miRNAs can participate in skeletal muscle regeneration by controlling the balance of pro- and anti-inflammatory factors in the skeletal muscle microenvironment.