miRNA Expression Profiling Uncovers a Role of miR-139-5p in Regulating the Calcification of Human Aortic Valve Interstitial Cells.

Calcific aortic valve disease (CAVD) is the most common structural heart disease, and the morbidity is increased with elderly population. Several microRNAs (miRNAs) have been identified to play crucial roles in CAVD, and numerous miRNAs are still waiting to be explored. In this study, the miRNA expression signature in CAVD was analyzed unbiasedly by miRNA-sequencing, and we found that, compared with the normal control valves, 152 miRNAs were upregulated and 186 miRNAs were downregulated in calcified aortic valves. The functions of these differentially expressed miRNAs were associated with cell differentiation, apoptosis, adhesion and immune response processes. Among downregulated miRNAs, the expression level of miR-139-5p was negatively correlated with the osteogenic gene RUNX2, and miR-139-5p was also downregulated during the osteogenic differentiation of primary human aortic valve interstitial cells (VICs). Subsequent functional studies revealed that miR-139-5p overexpression inhibited the osteogenic differentiation of VICs by negatively modulating the expression of pro-osteogenic gene FZD4 and CTNNB1. In conclusion, these results suggest that miR-139-5p plays an important role in osteogenic differentiation of VICs via the Wnt/ß-Catenin pathway, which may further provide a new therapeutic target for CAVD.

[Effect of arachidonic acid cytochrome P450ω hydroxylase Cyp4a14 gene knockout on skeletal muscle regeneration after injury].

The objective of this study was to explore the roles of arachidonic acid cytochrome P450ω hydroxylase CYP4A14 in skeletal muscle regeneration after injury. Wild-type (WT) control mice and Cyp4a14 knockout (A14-/-) mice were used to establish the muscle injury and regeneration model by intramuscular injection with cardiotoxin (CTX) on the tibial anterior (TA) muscle. The TA muscles were harvested at the time points of 0, 3, 5 and 15 days after injury. The changes in skeletal muscle regeneration and fibrosis were assessed by wheat germ agglutinin (WGA) staining and Sirius Red staining. Immunohistochemical staining was used to observe the expression of proliferation-related protein Ki-67 and macrophage marker protein Mac-2. The mRNA levels of regeneration and inflammation associated genes were analyzed by real-time PCR. The results showed that the cross-section area (CSA) of regenerated myofibers in A14-/- mice was significantly smaller (P < 0.05), while the percentage of fibrosis area was significantly higher than those in WT mice at 15 days after injury (P < 0.05). In A14-/- muscles, both the ratio of Ki-67 positive proliferating cells and the mRNA levels of differentiation associated genes Myod1 and Myog were significantly lower than those in WT muscles (P < 0.05). At 3 days after injury, the mRNA expression of inflammatory cells marker genes CD45 and CD11b and Mac-2 positive macrophages in A14-/- muscles were significantly lower than those in WT skeletal muscle (P < 0.05). Macrophages derived pro-regeneration cytokines IL-1ß, IGF-1 and SDF-1 were also significantly decreased in A14-/- muscles (P < 0.05). These results suggest that arachidonic acid cytochrome P450ω hydroxylase CYP4A14 plays a critical role in skeletal muscle regeneration after injury.

MicroRNA-200b-3p promotes endothelial cell apoptosis by targeting HDAC4 in atherosclerosis.

BACKGROUND: Epicardial adipose tissue (EAT) shares the same microcirculation with coronary arteries through coronary arteries branches, and contributes to the development of atherosclerosis. MicroRNAs (miRNAs) are involved in the formation of atherosclerosis. However, the alteration of miRNA profile in EAT during atherosclerosis is still uncovered. METHODS: The miRNA expression profiles of EAT from non-coronary atherosclerosis disease (CON, n = 3) and coronary atherosclerosis disease (CAD, n = 5) patients was performed to detect the differentially expressed miRNA. Then the expression levels of miRNA in other CON (n = 5) and CAD (n = 16) samples were confirmed by realtime-PCR. miR-200b-3p mimic was used to overexpress the miRNA in HUVECs. The apoptosis of HUVECs cells was induced by H2O2 and ox-LDL, and detected by Annexin V/PI Staining, Caspase 3/7 activity and the expression of BCL-2 and BAX. RESULTS: 250 miRNAs were differentially expressed in EAT from CAD patients, which were associated with metabolism, extracellular matrix and inflammation process. Among the top 20 up-regulated miRNAs, the expression levels of miR-200 family members (hsa-miR-200b/c-3p, miR-141-3p and miR-429), which were rich in endothelial cells, were increased in EAT from CAD patients significantly. Upregulation of miR-200 family members was dependent on the oxidative stress. The overexpression of miR-200b-3p could promote endothelial cells apoptosis under oxidative stress by targeting HDAC4 inhibition. CONCLUSIONS: Our study suggests that EAT derived miR-200b-3p promoted oxidative stress induced endothelial cells damage by targeting HDAC4, which may provide a new and promising therapeutic target for AS.

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.