Investigation of autophagy­related genes and immune infiltration in calcific aortic valve disease: A bioinformatics analysis and experimental validation.

The present study aimed to elucidate the role of autophagy-related genes (ARGs) in calcific aortic valve disease (CAVD) and their potential interactions with immune infiltration via experimental verification and bioinformatics analysis. A total of three microarray datasets (GSE12644, GSE51472 and GSE77287) were obtained from the Gene Expression Omnibus database, and gene set enrichment analysis was performed to identify the relationship between autophagy and CAVD. After differentially expressed genes and differentially expressed ARGs (DEARGs) were identified using CAVD samples and normal aortic valve samples, a functional analysis was performed, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses, protein-protein interaction network construction, hub gene identification and validation, immune infiltration and drug prediction. The results of the present study indicated a significant relationship between autophagy and CAVD. A total of 46 DEARGs were identified. GO and pathway enrichment analyses revealed the complex roles of DEARGs in regulating CAVD, including multiple gene functions and pathways. A total of 10 hub genes were identified, with three (SPP1, CXCL12 and CXCR4) consistently upregulated in CAVD samples compared with normal aortic valve samples in multiple datasets and experimental validation. Immune infiltration analyses demonstrated significant differences in immune cell proportions between CAVD samples and normal aortic valve samples, thus showing the crucial role of immune infiltration in CAVD development. Furthermore, therapeutic drugs were predicted that could target the identified hub genes, including bisphenol A, resveratrol, progesterone and estradiol. In summary, the present study illuminated the crucial role of autophagy in CAVD development and identified key ARGs as potential therapeutic targets. In addition, the observed immune cell infiltration and predicted autophagy-related drugs suggest promising avenues for future research and novel CAVD treatments.

The covalent NLRP3-inflammasome inhibitor Oridonin relieves myocardial infarction induced myocardial fibrosis and cardiac remodeling in mice.

BACKGROUND: Myocardial infarction (MI) triggers a strong inflammatory response that is associated with myocardial fibrosis and cardiac remodeling. Interleukin (IL)-1ß and IL-18 are key players in this response and are controlled by NLRP3-inflammatory bodies. Oridonin is a newly reported NLRP3 inhibitor with strong anti-inflammatory activity. We hypothesized that the covalent NLRP3 inhibitor Oridonin could reduce IL-1ß and IL-18 expression and ameliorate myocardial fibrosis after myocardial infarction in mice, improve poor heart remodeling, and preserve heart function. METHODS: Male C57BL/6 mice were subjected to left coronary artery ligation to induce MI and then treated with Oridonin (1, 3, or 6 mg/kg), MCC950 (10 mg/kg), CY-09 (5 mg/kg) or saline three times a week for two weeks. Four weeks after MI, cardiac function and myocardial fibrosis were assessed. In addition, myocardial expressions of inflammatory factors and fibrotic markers were analyzed by western blot, immunofluorescence, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction. RESULTS: Oridonin treatment preserved left ventricular ejection fraction and fractional shortening, and markedly limited the myocardial infarct size in treated mice. The myocardial fibrosis was lower in the 1 mg/kg group (15.98 ± 1.64)%, 3 mg/kg group (17.39 ± 2.45)%, and 6 mg/kg group (16.76 ± 3.06)% compared to the control group (23.38 ± 1.65)%. Moreover, similar with the results of Oridonin, MCC950 and CY-09 also preserved cardiac function and reduced myocardial fibrosis. The expression levels of NLRP3, IL-1ß and IL-18 were decreased in the Oridonin treatment group compared to non-treated group. In addition, myocardial macrophage and neutrophil influxes were attenuated in the Oridonin treated group. CONCLUSIONS: The covalent NLRP3-inflammasome inhibitor Oridonin reduces myocardial fibrosis and preserves cardiac function in a mouse MI model, which indicates potential therapeutic effect of Oridonin on acute MI patients.

Nanotubular TiO2 regulates macrophage M2 polarization and increases macrophage secretion of VEGF to accelerate endothelialization via the ERK1/2 and PI3K/AKT pathways.

Background: Macrophages play important roles in the immune response to, and successful implantation of, biomaterials. Titanium nanotubes are considered promising heart valve stent materials owing to their effects on modulation of macrophage behavior. However, the effects of nanotube-regulated macrophages on endothelial cells, which are essential for stent endothelialization, are unknown. Therefore, in this study we evaluated the inflammatory responses of endothelial cells to titanium nanotubes prepared at different voltages. Methods and results: In this study we used three different voltages (20, 40, and 60 V) to produce titania nanotubes with three different diameters by anodic oxidation. The state of macrophages on the samples was assessed, and the supernatants were collected as conditioned media (CM) to stimulate human umbilical vein endothelial cells (HUVECs), with pure titanium as a control group. The results indicated that titanium dioxide (TiO2) nanotubes induced macrophage polarization toward the anti-inflammatory M2 state and increased the expression of arginase-1, mannose receptor, and interleukin 10. Further mechanistic analysis revealed that M2 macrophage polarization controlled by the TiO2 nanotube surface activated the phosphatidylinositol 3-kinase/AKT and extracellular signal-regulated kinase 1/2 pathways through release of vascular endothelial growth factor to influence endothelialization. Conclusion: Our findings expanded our understanding of the complex influence of nanotubes in implants and the macrophage inflammatory response. Furthermore, CM generated from culture on the TiO2 nanotube surface may represent an integrated research model for studying the interactions of two different cell types and may be a promising approach for accelerating stent endothelialization through immunoregulation.

Biofunctionalization of decellularized porcine aortic valve with OPG-loaded PCL nanoparticles for anti-calcification.

Decellularized valve stents are widely used in tissue-engineered heart valves because they maintain the morphological structure of natural valves, have good histocompatibility and low immunogenicity. However, the surface of the cell valve loses the original endothelial cell coverage, exposing collagen and causing calcification and decay of the valve in advance. In this study, poly ε-caprolactone (PCL) nanoparticles loaded with osteoprotegerin (OPG) were bridged to a decellularized valve using a nanoparticle drug delivery system and tissue engineering technology to construct a new anti-calcification composite valve with sustained release function. The PCL nanoparticles loaded with OPG were prepared via an emulsion solvent evaporation method, which had a particle size of 133 nm and zeta potential of -27.8 mV. Transmission electron microscopy demonstrated that the prepared nanoparticles were round in shape, regular in size, and uniformly distributed, with an encapsulation efficiency of 75%, slow release in vitro, no burst release, no cytotoxicity to BMSCs, and contained OPG nanoparticles in vitro. There was a delay in the differentiation of BMSCs into osteoblasts. The decellularized valve modified by nanoparticles remained intact and its collagen fibers were continuous. After 8 weeks of subcutaneous implantation in rats, the morphological structure of the valve was almost complete, and the composite valve showed anti-calcification ability to a certain extent.