Angiopoietin-2 induces angiogenesis via exosomes in human hepatocellular carcinoma.

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. Video Abstract.

Vps4A functions as a tumor suppressor by regulating the secretion and uptake of exosomal microRNAs in human hepatoma cells.

UNLABELLED: The deregulation of microRNAs (miRNAs) plays an important role in human hepatocarcinogenesis. In this study, we highlight exosomes as mediators involved in modulating miRNA profiles in hepatocellular carcinoma (HCC) cells. First, we examined the different miRNA expression profiles in HCC cells and HCC cell-derived exosomes. Next, coculture experiments indicated that HCC cell-derived exosomes promoted the cell growth, migration, and invasion of HCC cells and had the ability to shuttle miRNAs to recipient cells. Further, our data showed that Vps4A, a key regulator of exosome biogenesis, was frequently down-regulated in HCC tissues. The reduction of Vps4A in HCC tissues was associated with tumor progression and metastasis. In vitro studies revealed that Vps4A repressed the growth, colony formation, migration, and invasion of HCC cells. We further investigated the role and involvement of Vps4A in suppressing the bioactivity of exosomes and characterized its ability to weaken the cell response to exosomes. By small RNA sequencing, we demonstrated that Vps4A facilitated the secretion of oncogenic miRNAs in exosomes as well as accumulation and uptake of tumor suppressor miRNAs in cells. A subset of Vps4A-associated miRNAs was identified. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that the phosphatidylinositol-3-kinase/Akt signaling pathway was the most likely candidate pathway for modulation by these miRNAs. Indeed, we proved that the phosphatidylinositol-3-kinase/Akt pathway was inactivated by Vps4A overexpression. CONCLUSION: Exosome-mediated miRNA transfer is an important mechanism of self-modulation of the miRNA expression profiles in HCC cells, and Vps4A may function as a tumor suppressor, which utilizes exosomes as mediators to regulate the secretion and uptake of miRNAs in hepatoma cells; these observations provide new insights into the development of HCC.

Anticancer drugs cause release of exosomes with heat shock proteins from human hepatocellular carcinoma cells that elicit effective natural killer cell antitumor responses in vitro.

Failure of immune surveillance related to inadequate host antitumor immune responses has been suggested as a possible cause of the high incidence of recurrence and poor overall survival outcome of hepatocellular carcinoma. The stress-induced heat shock proteins (HSPs) are known to act as endogenous "danger signals" that can improve tumor immunogenicity and induce natural killer (NK) cell responses. Exosome is a novel secretory pathway for HSPs. In our experiments, the immune regulatory effect of the HSP-bearing exosomes secreted by human hepatocellular carcinoma cells under stress conditions on NK cells was studied. ELISA results showed that the production of HSP60, HSP70, and HSP90 was up-regulated in both cell lines in a stress-specific manner. After exposure to hepatocellular carcinoma cell-resistant or sensitive anticancer drugs (hereafter referred to as "resistant" or "sensitive" anticancer drug), the membrane microvesicles were actively released by hepatocellular carcinoma cells, differing in their ability to present HSPs on the cell surface, which were characterized as exosomes. Acting as a decoy, the HSP-bearing exosomes efficiently stimulated NK cell cytotoxicity and granzyme B production, up-regulated the expression of inhibitory receptor CD94, and down-regulated the expression of activating receptors CD69, NKG2D, and NKp44. Notably, resistant anticancer drugs enhanced exosome release and generated more exosome-carried HSPs, which augmented the activation of the cytotoxic response. In summary, our findings demonstrated that exosomes derived from resistant anticancer drug-treated HepG2 cells conferred superior immunogenicity in inducing HSP-specific NK cell responses, which provided a clue for finding an efficient vaccine for hepatocellular carcinoma immunotherapy.

Functional distinction of rat liver natural killer cells from spleen natural killer cells under normal and acidic conditions in vitro.

BACKGROUND: The microenvironment within solid tumors has often been shown to exhibit an acidic extracellular pH. Although the morphologic and functional differences in natural killer (NK) cells of the liver and spleen have been reported previously under physiological conditions, the difference under acidic conditions is still unclear. This study was to investigate the differences in the morphological and functional characteristics between rat liver and spleen NK cells under normal and acidic conditions in vitro. METHODS: Liver and spleen NK cells were isolated and purified from Sprague-Dawley rats by density gradient centrifugation and the Dynabeads(®) FlowComp(TM) Flexi system, and stimulated for 4 days with or without IL-2 or treated with low pH or control for different times. Morphology was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cell death and proliferation assays were performed by flow cytometry, IFN-gamma production was tested by ELISA, and cytotoxic activity was evaluated by lactate dehydrogenase (LDH) release assay. RESULTS: Liver NK cells had significantly higher levels of cytotoxic activity than spleen NK cells under normal and acidic conditions, and the maximum difference was observed at pH 5.6. Further analysis revealed that the cytotoxic activity of NK cells was correlated with morphology, cell death, proliferative activity and IFN-gamma production. By TEM, liver NK cells contained a greater number of electron-dense granules per cell at pH 5.6. Moreover, a modest elevation of cell death and reduction of proliferation of liver NK cells occurred within a range of 5.6-7.2. Interestingly, an acidic extracellular pH only marginally, and not significantly, suppressed IFN-gamma production by liver NK cells. CONCLUSION: The sharp morphological and functional differences shown by the two types of NK cells in vitro indicate that liver NK cells are unexpectedly resistant to pH shock.

Spontaneous expulsion of a duodenal lipoma after endoscopic biopsy: A case report.

BACKGROUND: Gastrointestinal (GI) lipomas are benign submucosal tumors of mature adipocytes that arise mainly in the colon and stomach, sometimes in the ileum and jejunum, and rarely in the duodenum. Patients with symptomatic lipomas require endoscopic or surgical treatment. Spontaneous expulsion of lipomas after biopsy is a rare condition that has limited case reports. CASE SUMMARY: A 56-year-old man presented to our hospital with intermittent postprandial epigastric fullness. Esophagogastroduodenoscopy (EGD) revealed a 10-mm soft yellowish submucosal lesion with the "pillow sign," located in the second portion of duodenum. Endoscopic ultrasonography (EUS) using a 12-MHz catheter probe showed a hyperechoic, homogenous, and round solid lesion (OLYMPUS EUS EU-ME2, UM-DP12-25R, 12-MHz radial miniprobe, Olympus Corporation, Tokyo, Japan). Deep biopsy was performed using the bite-on-bite technique with forceps. Histological examination was compatible with submucosal lipoma. The lesion spontaneously expelled 12 d after the biopsy. Follow-up EUS performed after 2 mo confirmed this condition. CONCLUSION: Deep biopsy could lead to spontaneous GI lipoma expulsion. This might be the first step in lipoma diagnosis and treatment.

Donor liver natural killer cells alleviate liver allograft acute rejection in rats.

BACKGROUND: Liver enriched natural killer (NK) cells are of high immune activity. However, the function of donor liver NK cells in allogeneic liver transplantation (LTx) remains unclear. METHODS: Ten Gy of whole body gamma-irradiation (WBI) from a 60Co source at 0.6 Gy/min was used for depleting donor-derived leukocytes, and transfusion of purified liver NK cells isolated from the same type rat as donor (donor type liver NK cells, dtlNKs) through portal vein was performed immediately after grafting the irradiated liver. Post-transplant survival observation on recipients and histopathological detection of liver grafts were adoptive to evaluate the biological impact of donor liver NK cells on recipients' survival in rat LTx. RESULTS: Transfusion of dtlNKs did not shorten the survival time among the recipients of spontaneous tolerance model (BN to LEW rat) after rat LTx, but prolonged the liver graft survival among the recipients depleted of donor-derived leukocytes in the acute rejection model (LEW to BN rat). Compared to the recipients in the groups which received the graft depleted of donor-derived leukocytes, better survival and less damage in the allografts were also found among the recipients in the two different strain combinations of liver allograft due to transfusion of dtlNKs. CONCLUSIONS: Donor liver NK cells alone do not exacerbate liver allograft acute rejection. Conversely, they can alleviate it, and improve the recipients' survival.

Evaluation on two types of paramyosin vaccines for the control of Haemaphysalis longicornis infestations in rabbits.

BACKGROUND: Haemaphysalis longicornis is an obligate hematophagous ectoparasite that transmits a variety of pathogens causing life-threatening diseases in humans and animals. Paramyosin (Pmy) is not only an invertebrate-specific myofibrillar protein but also an important immunomodulatory protein. Therefore, it is one of the ideal candidate antigens for vaccines. METHODS: We conducted two vaccine trials to evaluate the protective efficacy of Pmy recombinant protein (rPmy) and peptide vaccine (KLH-LEE). Each rabbit was immunized with three doses of rPmy or KLH-LEE adjuvanted with Freund's complete/incomplete at 500 µg/dose at 2-week intervals before challenge with 40 female H. longicornis/rabbit. PBS plus adjuvant, Trx or KLH was used as control group. The antibodies of rabbits were detected by ELISA. Then, female ticks were fed on the rabbits until detachment. RESULTS: ELISA results showed that both vaccines induced rabbits to produce antibodies. Compared with the Trx group, the engorgement weight, oviposition and hatchability of the rPmy group decreased by 8.87%, 26.83% and 38.86%, respectively. On the other hand, engorgement weight, oviposition and hatchability of female ticks in the KLH-LEE group correspondingly resulted in 27.03%, 53.15% and 38.40% reduction compared with that of the KLH group. Considering the cumulative effect of vaccination on the evaluated parameters, results showed 60.37% efficacy of the rPmy vaccine formulation and 70.86% efficacy in the KLH-LEE group. CONCLUSIONS: Pmy and particularly epitope LEE have potential for further development of an effective candidate vaccine to protect the host against tick infection. GRAPHIC ABSTARCT.

Hepatocyte infusions improve the results of liver transplantation in rats.

BACKGROUND/AIMS: The results of liver transplantation were influenced by miscellaneous factors. The aim was the notion that hepatocyte infusions might improve results of liver transplantation in rats. METHODOLOGY: Survival, serum analytes, FasL production, and histologic degree of rejection of transplanted rats pretreated with hepatocyte infusions (Hepatocyte) was compared to that of rats pretreated with either bone marrow cell infusions (Marrow) or saline infusions (Control). RESULTS: Hepatocyte rats had longer median survivals, less biochemical evidence of liver damage, more albumin production, a lower degree of histologic rejection, and increased FasL production than did either Marrow or Control rats (p < 0.05 for each analysis), which did not differ from one another (p > 0.05 for each analysis). CONCLUSIONS: Hepatocyte infusions improve the results of liver transplantation in rats.

Medical expenditure for esophageal cancer in China: a 10-year multicenter retrospective survey (2002-2011).

BACKGROUND: Esophageal cancer is associated with substantial disease burden in China, and data on the economic burden are fundamental for setting priorities in cancer interventions. The medical expenditure for the diagnosis and treatment of esophageal cancer in China has not been fully quantified. This study aimed to examine the medical expenditure of Chinese patients with esophageal cancer and the associated trends. METHODS: From 2012 to 2014, a hospital-based multicenter retrospective survey was conducted in 37 hospitals in 13 provinces/municipalities across China as a part of the Cancer Screening Program of Urban China. For each esophageal cancer patient diagnosed between 2002 and 2011, clinical information and expense data were extracted by using structured questionnaires. All expense data were reported in Chinese Yuan (CNY; 1 CNY = 0.155 USD) based on the 2011 value and inflated using the year-specific health care consumer price index for China. RESULTS: A total of 14,967 esophageal cancer patients were included in the analysis. It was estimated that the overall average expenditure per patient was 38,666 CNY, and an average annual increase of 6.27% was observed from 2002 (25,111 CNY) to 2011 (46,124 CNY). The average expenditures were 34,460 CNY for stage I, 39,302 CNY for stage II, 40,353 CNY for stage III, and 37,432 CNY for stage IV diseases (P < 0.01). The expenditure also differed by the therapy type, which was 38,492 CNY for surgery, 27,933 CNY for radiotherapy, and 27,805 CNY for chemotherapy (P < 0.05). Drugs contributed to 45.02% of the overall expenditure. CONCLUSIONS: These conservative estimates suggested that medical expenditures for esophageal cancer in China substantially increased in the last 10 years, treatment for early-stage esophageal cancer costs less than that for advanced cases, and spending on drugs continued to account for a considerable proportion of the overall expenditure.

Effect of propofol on generation of inflammatory mediator of monocytes.

OBJECTIVE: To evaluate the effect of propofol with different concentrations on the expression of inflammatory mediators of interleukin and tumor-necrosis factor-α (TNF-α) by stimulating the mouse primary monocytes and human monocytic cell line with lipopolysaccharide (LPS) and also discuss the effect of propofol on the secretion of inflammatory mediator and its possible molecular mechanism. METHODS: The mononuclear cells of mouse spleen were separated and then purified to obtain the primary monocytes. The dose-effect relationship of production of pro-inflammatory cytokines by monocytes which were stimulated by LPS, namely the monocytes were stimulated by the dose of 0-500 ng/mL for 24 h. ELISA was employed to detect the concentration of IL-6, IL-8 and TNF-α. The effect of propofol on the secretion of above pro-inflammatory cytokines by the monocytes was observed. Cells were divided into the control group, the 0.1% DMSO group, the LPS group and the treatment group with LPS + different dose of propofol (propofol 1-100 µg/mL). ELISA was employed to detect the concentration of IL-6, IL-8 and TNF-α. The change in the expression of important signaling molecules in Toll-like receptor and NF-κB signaling pathway was detected after THP-1 cells were treated with propofol. RESULTS: The concentration of TNF-α was (3863 ± 153) pg/mL after 12 h of stimulation by LPS and then its concentration was decreased gradually. But the concentration of IL-6 and IL-8 was relatively high after 24 h of stimulation by LPS, (5627 ± 330) pg/mL and (1626 ± 200) pg/mL, respectively. The propofol could inhibit the expression of IL-6, IL-8 and TNF-α caused by LPS. After the intervention treatment of 50 µg/mL propofol, the concentration of IL-6, IL-8 and TNF-α was significantly decreased (P < 0.01). CONCLUSIONS: The propofol can inhibit the expression of TLR-4 and NF-κB to inhibit the activation of p38 and the expression of pro-inflammatory cytokines.

High incidence of biliary complications in rat liver transplantation: can we avoid it?

AIM: To investigate how to reduce the incidence of biliary complications in rat orthotopic liver transplantation. METHODS: A total of 165 male Wistar rats were randomly divided into three groups: Group A, orthotropic liver transplantation with modified "two-cuff" technique; Group B, bile duct was cut and reconstructed without transplantation; and Group C, only laparotomy was performed. Based on the approaches used for biliary reconstruction, Group A was divided into two sub-groups:A1 (n = 30), duct-duct reconstruction, and A2 (n = 30), duct-duodenum reconstruction. To study the influence of artery reconstruction on bile duct complication, Group B was divided into four sub-groups: B1 (n = 10), duct-duct reconstruction with hepatic artery ligation, B2 (n = 10), duct-duct reconstruction without hepatic artery ligation, B3 (n = 10), duct-duodenum reconstruction with hepatic artery ligation, and B4 (n = 10), duct-duodenum reconstruction without hepatic artery ligation. The samples were harvested 14 d after operation or at the time when significant biliary complication was found. RESULTS: In Group A, the anhepatic phase was 13.7 ± 1.06 min, and cold ischemia time was 50.5 ± 8.6 min. There was no significant difference between A1 and A2 in the operation duration. The time for biliary reconstruction was almost the same among all groups. The success rate for transplantation was 98.3% (59/60). Significant differences were found in the incidence of biliary complications in Groups A (41.7%), B (27.5%) and C (0%). A2 was more likely to have biliary complications than A1 (50% vs 33.3%). B3 had the highest incidence of biliary complications in Group B. CONCLUSION: Biliary complications are almost inevitable using the classical "two cuff" techniques, and duct-duodenum reconstruction is not an ideal option in rat orthotopic liver transplantation.