Hepatocellular carcinoma (HCC) patients suffer from a poor survival rate and a high incidence of postoperative recurrence. The hepatic microenvironment plays a significant role in the initiation, progression, and recurrence of HCC; however, the causal mechanisms of these phenomena are unclear. Given the predominant underlying fibrotic and cirrhotic conditions of the liver prone to HCC and its recurrence, alterations of components of the inflammatory milieu have been suggested as factors that promote HCC development. In particular, activated hepatic stellate cells (A‐HSCs), which play a key role in liver fibrosis and cirrhosis, have been suggested as contributors to the HCC‐prone microenvironment. Here, we have identified and validated an A‐HSC‐specific gene expression signature among nontumor tissues of 319 HCC patients that is significantly and independently associated with HCC recurrence and survival. Peritumoral, rather than tumor tissue‐related, A‐HSC‐specific gene expression is associated with recurrence and poor survival. Analyses of A‐HSC‐specific gene signatures and further immunohistochemical validation in an additional 143 HCC patients have revealed that A‐HSCs preferentially affect monocyte populations, shifting their gene expression from an inflammatory to an immunosuppressive signature. In addition, the interaction between A‐HSCs and monocytes induces protumorigenic and progressive features of HCC cells by enhancing cell migration and tumor sphere formation. Conclusion: A‐HSCs play a significant role in promoting HCC progression through interaction with and alteration of monocyte activities within the liver microenvironment; thus, disrupting the interactions and signaling events between the inflammatory milieu and components of the microenvironment may be useful therapeutic strategies for preventing HCC tumor relapse. (Hepatology 2015;62:481–495

The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.

Serum small extracellular vesicles (sEVs) have recently drawn considerable interest because of the diagnostic and therapeutic potential of their miRNAs content. However, the characteristics of human, mouse and rat serum sEVs and their differences in small RNA contents are still unknown. In this study, through nanoparticle tracking analysis and small RNA sequencing, we found that human, rat, and mouse serum sEVs exhibited distinct sizes and particle numbers as well as small RNA contents. Serum sEVs contained not only abundant miRNAs but also a large number of tRNA fragments. Most serum miRNAs existed both inside and outside of sEVs but were enriched in sEVs. Common serum sEV miRNAs (188 miRNAs) and species-specific serum sEV miRNAs (265, 58, and 159 miRNAs, respectively) were identified in humans, rats, or mice. The serum sEVs contained miRNAs from tissues and organs throughout the body, with blood cells as the main contributors. In conclusion, our findings confirmed the rationality of exploring serum sEV miRNAs as noninvasive diagnostic markers and revealed great differences in serum sEV small RNAs between humans, rats, and mice. Inadequate attention to these differences and the contribution of blood cells to serum sEV miRNAs could hinder the clinical translation of basic studies.

Elucidation of the molecular events underlying hepatic stellate cell (HSC) activation is an essential step toward understanding the biological properties of HSC and clarifying the potential roles of HSCs in liver fibrosis and other liver diseases, including hepatocellular carcinoma. High-throughput comparative proteomic analysis based on isobaric tags for relative and absolute quantitation (iTRAQ) labeling combined with online two-dimensional nanoscale liquid chromatography and tandem mass spectrometry (2D nano-LC-MS/MS) were performed on an in vitro HSC activation model to obtain a comprehensive view of the protein ensembles associated with HSC activation. In total, 2,417 proteins were confidently identified (false discovery rate <1%), of which 2,322 proteins were quantified. Compared with quiescent HSCs, 519 proteins showed significant differences in activated HSCs (≥3.0-fold). Bioinformatics analyses using Ingenuity Pathway Analysis revealed that the 319 up-regulated proteins represented multiple cellular functions closely associated with HSC activation, such as extracellular matrix synthesis and proliferation. In addition to the well-known markers for HSC activation, such as α-smooth muscle actin and collagen types 1 and 3, some novel proteins potentially associated with HSC activation were identified, while the 200 down-regulated proteins were primarily related to immune response and lipid metabolism. Most intriguingly, the top biological function, top network, and top canonical pathway of down-regulated proteins were all involved in immune responses. The expression and/or biological function of a set of proteins were properly validated, especially Bcl2-associated athanogene 2, BAG3, and B7H3. Conclusion: The present study provided the most comprehensive proteome profile of rat HSCs and some novel insights into HSC activation, especially the suppressed immune response. (HEPATOLOGY 2012;56:332–349)