Arterial calcification is a major complication of cardiovascular disease. Oestrogen replacement therapy in postmenopausal women is associated with lower levels of coronary artery calcification, but its mechanism of action remains unclear. Here, we show that oestrogen inhibits the osteoblastic differentiation of vascular smooth muscle cells (VSMCs) in vitro and arterial calcification in vivo by promoting autophagy. Through electron microscopy, GFP–LC3 redistribution, and immunofluorescence analyses as well as measurement of the expression of the autophagosome marker light-chain I/II (LC3I/II) and autophagy protein 5 (Atg5), we show that autophagy is increased in VSMCs by oestrogen in vitro and in vivo. The inhibitory effect of oestrogen on arterial calcification was counteracted by 3-methyladenine (3MA) or knockdown of Atg5 and was increased by rapamycin. Furthermore, the inhibitory effect of oestrogen on arterial calcification and the degree of autophagy induced by oestrogen were blocked by a nonselective oestrogen receptor (ER) antagonist (ICI 182780), a selective oestrogen receptor alpha (ERα) antagonist (MPP), and ERα-specific siRNA. Our data indicate that oestrogen inhibits the osteoblastic differentiation of VSMCs by promoting autophagy through the ERα signalling pathway in vitro and arterial calcification in vivo by increasing autophagy. Our findings provide new insights into the mechanism by which oestrogen contributes to vascular calcification in vitro and in vivo.

Arterial calcification is a common cardiovascular disease that initiates from a process of osteoblastic differentiation of vascular smooth muscle cells (VSMCs). Accumulating evidence has demonstrated that microRNAs play an important role in regulating arterial calcification. miR-204 was significantly downregulated in calcified human renal arteries from patients with uremia; calcified arteries of mice, due to 5/6 nephrectomy with a high-phosphate diet (5/6 NTP); and in VSMCs induced by high phosphate concentration. The overexpression of miR-204 alleviated the osteoblastic differentiation of VSMCs. Bisulphite sequencing PCR revealed that CpG sites upstream of miR-204 DNA were hypermethylated in calcified VSMCs; in calcified arteries of mice, due to 5/6 NTP; and in calcified renal artery tissues from patients with uremia. Moreover, increased DNMT3a resulted in the hypermethylation of miR-204 in high phosphate concentration-induced VSMCs, whereas 5-aza-2'-deoxycytidine could restore the expression of miR-204 in high phosphate concentration-induced VSMCs. Moreover, we found that DNMT3a was the target of miR-204, and the methylation ratio of miR-204 was decreased significantly, meaning that the expression of miR-204 was restored when DNMT3a was knocked down by using DNMT3a small interfering RNA, resulting in abrogation of the effect of high phosphate concentration on VSMC calcification. The progress of arterial calcification is regulated by the miR-204/DNMT3a regulatory circuit.

SerpinE2 is a member of the Serpins family, which could inhibit serine protease and promote tumor progression, particularly in tumor metastasis. However, at present, its role in the progression of osteosarcoma has not been determined. The present study analyzed the expression profiles of SerpinE2 in cancer tissues, including tissues from osteosarcoma of different stages. Higher expression of SerpinE2 was shown in osteosarcoma tissues, particularly in tissue from patients with metastasis and a tumor-node-metastasis stage II‑III. Following chemotherapy, the SerpinE2 expression levels were shown to be higher than those at diagnosis. Cell proliferation and colony formation were increased after transfection with SerpinE2 over‑expression vector. Additionally, drug resistance to bortezomib and doxorubicin treatment following SerpinE2 transfection was analyzed. MG‑63 and SAOS‑2 cells showed less sensitivity following transfection with SerpinE2. The cell cycle‑related genes, cyclin‑dependent kinase (CDK)4 and cyclin D1 were positively correlated with SerpinE2 expression in patient‑derived tissue and in osteosarcoma cells. Finally, the high expression of SerpinE2 contributes to poor survival rates in patients with osteosarcoma. In conclusion, high expression of SerpinE2 in osteosarcoma stimulates cell proliferation, promotes drug‑resistance, and results in poor survival by regulating CDK4 and cyclin D1. Thus, SerpinE2 could be a potential target for treatment of patients with osteosarcoma.

The present study aimed to investigate the effect of co‑culture of fibroblast‑like synoviocytes (FLS) with human umbilical cord‑mesenchymal stem cells (UC‑MSCs) on rheumatoid arthritis (RA) and to understand the mechanisms that mediate the induced changes. FLS and UC‑MSCs were isolated and cultured individually, FLS were then cultured with or without UC‑MSCs. The phenotype of UC‑MSCs was analyzed prior to co‑culture. The UC‑MSCs were successfully isolated and expanded, and exhibited a fibroblast‑like morphology. Enzyme‑linked immunosorbent assay (ELISA) and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) were performed to determine the expression levels of interleukin (IL)‑1β, IL‑6, and chemokine (C‑C motif) ligand (CCL)‑2. The cell apoptosis rate was determined by flow cytometry. Furthermore, the RNAs of aggrecan and collagen type II were isolated and assessed in a chondrogenesis assay following co‑culture for 7, 14, 21 and 28 days. Protein expression levels of apoptosis‑related proteins, including B‑cell lymphoma (Bcl‑2), Bcl‑2‑associated X protein, p53 and phospho (p)‑AKT, and growth differentiation factor‑5 were analyzed by western blotting. ELISA and qRT‑PCR demonstrated that compared with FLS cultured alone, co‑culture with UC‑MSCs significantly downregulates the expression levels of IL‑1β, IL‑6 and CCL‑2. Additionally, the percentage of apoptotic cells was significantly increased in the co‑cultured cells (P<0.05), and the relative RNAs levels of aggrecan and collagen type II were increased compared with FLS alone. Furthermore, the expression levels of Bcl‑2 (P<0.05) and p‑AKT (P<0.05) were significantly decreased, whereas, p53 (P=0.001), Bax (P<0.01) and GDF‑5 (P<0.01) were increased by co‑culture of FLS with UC‑MSCs compared with FLS alone. In conclusion, co‑culture of FLS with UC‑MSCs may be important and clinically useful for the treatment of RA by inhibiting the expression of pro‑inflammatory mediators, inducing apoptosis and promoting chondrogenesis.