Background: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is a highly vascularized solid tumor. Angiopoietin-2 (ANGPT2) has been described as an attractive target for antiangiogenic therapy. Exosomes are small extracellular vesicles secreted by most cell types and contribute to cell-to-cell communication by delivering functional cargo to recipient cells. The expression of ANGPT2 in tumor-derived exosomes remains unknown. Methods: We detected the ANGPT2 expression in HCC-derived exosomes by immunoblotting, enzyme-linked immunosorbent assay and immunogold labeling, then observed exosomal ANGPT2 internalization and recycling by confocal laser scanning microscopy, co-immunoprecipitation and immunoblotting. We used two HCC cell lines (Hep3B and MHCC97H) to overexpress ANGPT2 by lentivirus infection or knockdown ANGPT2 by the CRISPR/Cas system, then isolated exosomes to coculture with human umbilical vein endothelial cells (HUVECs) and observed the angiogenesis by Matrigel microtubule formation assay, transwell migration assay, wound healing assay, cell counting kit-8 assay, immunoblotting and in vivo tumorigenesis assay. Results: We found that HCC-derived exosomes carried ANGPT2 and delivered it into HUVECs by exosome endocytosis, this delivery led to a notable increase in angiogenesis by a Tie2-independent pathway. Concomitantly, we observed that HCC cell-secreted exosomal ANGPT2 was recycled by recipient HUVECs and might be reused. In addition, the CRISPR-Cas systems to knock down ANGPT2 significantly inhibited the angiogenesis induced by HCC cell-secreted exosomal ANGPT2, and obviously suppressed the epithelial-mesenchymal transition activation in HCC. Conclusions: Taken together, these results reveal a novel pathway of tumor angiogenesis induced by HCC cell-secreted exosomal ANGPT2 that is different from the classic ANGPT2/Tie2 pathway. This way may be a potential therapeutic target for antiangiogenic therapy.

Rationale: Cardiomyocyte apoptosis critically contributes to ischemic heart failure (IHF) progression. While the endosome-lysosome system governs cellular homeostasis, the functional significance of its master regulator RAB7 in cardiac pathophysiology remains unexplored.

Background: Hypertrophic cardiomyopathy is an independent risk factor for heart failure. Citrus reticulata (C. reticulata) is a traditional Chinese medicine with a variety of chemical components and pharmacological effects. The mechanisms of C. reticulata for treating hypertrophic cardiomyopathy are still unclear.Methods: In this study, we used network pharmacology techniques combined with bioinformatics analysis and identified the active ingredient in C. reticulata to protect against hypertrophic cardiomyopathy. We analyzed the Gene Expression Omnibus (GEO) database from human heart tissue with hypertrophic cardiomyopathy to reveal the potential targets. Finally, molecular docking and in vitro validation were used to reveal the binding of the potential targets and the main active component of C. reticulata.Results: Our results revealed that there are five main active ingredients of C. reticulata (nobiletin, naringenin, sitosterol, 5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl) chroman-4-one, and citromitin). By analyzing the intersecting genes between C. reticulata and hypertrophic cardiomyopathy, 40 hub genes were obtained. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the responses to oxidative stress and fatty acids were the main pathways for C. reticulata to act against hypertrophic cardiomyopathy. The protein–protein interaction analysis results showed that the main active ingredients of C. reticulata were nobiletin and naringenin, while peroxisome proliferator-activated receptors (PPAR)α might be the potential targets of C. reticulata in treating hypertrophic cardiomyopathy. The molecular docking results showed that the main active ingredient, nobiletin, could bind to PPARα with a strong hydrogen-bonding interaction force. In vitro results validated that nobiletin might directly bind to PPARα, thereby increasing the expression of lipid metabolism-related genes and relieving hypertrophic responses of cardiomyocytes.Conclusion: The nuclear receptor PPARα might be the potential endogenous receptor of the active ingredients of C. reticulata.