Reconstruction of hepatic stellate cell‐incorporated liver capillary structures in small hepatocyte tri‐culture using microporous membranes

In liver sinusoids, hepatic stellate cells (HSCs) locate the outer surface of microvessels to form a functional unit with endothelia and hepatocytes. To reconstruct functional liver tissue in vitro, formation of the HSC‐incorporated sinusoidal structure is essential. We previously demonstrated capillary formation of endothelial cells (ECs) in tri‐culture, where a polyethylene terephthalate (PET) microporous membrane was intercalated between the ECs and hepatic organoids composed of small hepatocytes (SHs), i.e. hepatic progenitor cells, and HSCs. However, the high thickness and low porosity of the membranes limited heterotypic cell–cell interactions, which are essential to form HSC–EC hybrid structures. Here, we focused on the effective use of the thin and highly porous poly( d , l ‐lactide‐co‐glycolide) (PLGA) microporous membranes in SH–HSC–EC tri‐culture to reconstruct the HSC‐incorporated liver capillary structures in vitro. First, the formation of EC capillary‐like structures was induced on Matrigel‐coated PLGA microporous membranes. Next, the membranes were stacked on hepatic organoids composed of small SHs and HSCs. When the pore size and porosity of the membranes were optimized, HSCs selectively migrated to the EC capillary‐like structures. This process was mediated in part by platelet‐derived growth factor (PDGF) signalling. In addition, the HSCs were located along the outer surface of the EC capillary‐like structures with their long cytoplasmic processes. In the HSC‐incorporated capillary tissues, SHs acquired high levels of differentiated functions, compared to those without ECs. This model will provide a basis for the construction of functional, thick, vascularized liver tissues in vitro. Copyright © 2012 John Wiley & Sons, Ltd.     

Histological and Histometric Evaluation of the Liver in Astyanax Bimaculatus (Teleostei: Characidae), Exposed to Different Concentrations of an Organochlorine Insecticide

To investigate possible morphological changes to the liver tissue of lambaris, Astyanax bimaculatus (Linnaeus, 1758), females were exposed to treatments of sublethal concentrations of the insecticide Thiodan^® for 96 hr. Treatments included three sublethal concentrations of 1.15, 2.3, and 5.6 μg L^?1 of Thiodan^® and a control group without insecticide. The action of Thiodan^® at sublethal concentrations did not affect the morphological structure of the liver as a whole, but changes in isolated locations of the hepatic parenchyma were observed. Glycogen depletion, nuclear and cytoplasmic deformation, nuclear and cytoplasmic hypertrophy, hyperemia, and cellular degeneration in liver cells at the different concentrations studied were recorded. These observed changes in the livers were greater in groups exposed to Thiodan^® in comparison to the control group. Furthermore, there was a change in the diameter of the nuclei and cytoplasm of hepatocytes in the different treatments. The groups exposed to Thiodan^® also exhibited a larger number of hepatocyte nuclei and a reduction in the amount of cytoplasm. We conclude that for the exposure period and concentrations of Thiodan^® analyzed, the morphology of hepatic tissue had a cellular adaptive response. Anat Rec, 298:1754–1764, 2015. © 2015 Wiley Periodicals, Inc.     

气道内滴注肝细胞生长因子对野百合碱诱导肺动脉高压大鼠的干预研究

目的 通过气道内滴注腺病毒转染肝细胞生长因子（adenovirus hepatocyte growth factor,Ad-HGF）干预野百合碱（monocrotaline,MCT）诱导的肺动脉高压（pulmonary hypertension,PAH）模型大鼠,观察Ad-HGF干预对平均肺动脉压（mean pulmonary artery pressure,mPAP）等指标的影响.方法 选用健康雄性SD大鼠40只,随机[采用随机单位（区）组设计方法]分为4组：正常对照组（normal,NOR组）10只,MCT诱导PAH组（PAH组）10只、单次和重复肝细胞生长因子（hepatocyte growth factor,HGF）干预治疗组（HGF和THGF组）各10只.NOR组和PAH组：气道内滴注0.2 mL磷酸盐缓冲液;HGF组和THGF组：气道内滴注0.2 mL Ad-HGF 1次和一周后重复一次共2次.再饲养两周后,测定各组大鼠的肺动脉压,计算右心室肥厚指数;苏木素伊红染色观察肺动脉管壁等;酶联免疫吸附（ELISA）法测定肺组织匀浆中HGF浓度.结果 与PAH组大鼠比较,HGF组和THGF组大鼠的mPAP、右心室肥厚指数、肺动脉管壁指数和面积指数明显降低,肺苏木素伊红染色肺小动脉管壁厚度减少,管腔面积增大,血管周围炎症细胞浸润减轻,肺组织匀浆HGF浓度明显升高,差异有统计学意义（P＜0.05）,且低于NOR组水平.结论 经气道内滴注转染Ad-HGF,能明显降低但不能完全逆转MCT诱导PAH大鼠的mPAP,减少肺小动脉管壁厚度,减轻右心室肥厚程度,从而达到延缓PAH进程的作用.     

Destruction of the hepatocyte junction by intercellular invasion of Leptospira causes jaundice in a hamster model of Weil's disease

Weil's disease, the most severe form of leptospirosis, is characterized by jaundice, haemorrhage and renal failure. The mechanisms of jaundice caused by pathogenic Leptospira remain unclear. We therefore aimed to elucidate the mechanisms by integrating histopathological changes with serum biochemical abnormalities during the development of jaundice in a hamster model of Weil's disease. In this work, we obtained three‐dimensional images of infected hamster livers using scanning electron microscope together with freeze‐cracking and cross‐cutting methods for sample preparation. The images displayed the corkscrew‐shaped bacteria, which infiltrated the Disse's space, migrated between hepatocytes, detached the intercellular junctions and disrupted the bile canaliculi. Destruction of bile canaliculi coincided with the elevation of conjugated bilirubin, aspartate transaminase and alkaline phosphatase levels in serum, whereas serum alanine transaminase and γ‐glutamyl transpeptidase levels increased slightly, but not significantly. We also found in ex vivo experiments that pathogenic, but not non‐pathogenic leptospires, tend to adhere to the perijunctional region of hepatocyte couplets isolated from hamsters and initiate invasion of the intercellular junction within 1 h after co‐incubation. Our results suggest that pathogenic leptospires invade the intercellular junctions of host hepatocytes, and this invasion contributes in the disruption of the junction. Subsequently, bile leaks from bile canaliculi and jaundice occurs immediately. Our findings revealed not only a novel pathogenicity of leptospires, but also a novel mechanism of jaundice induced by bacterial infection.     

Acetaminophen‐induced hepatocyte injury: C2‐ceramide and oltipraz intervention,hepatocyte nuclear factor 1 and glutathione S‐transferase A1 changes

Acetaminophen (APAP) is an antipyretic and analgesic, which is commonly associated with drug‐induced hepatic injury. C2‐ceramide plays a key role in mediating cell life activities, and oltipraz was extensively studied as a cancer chemopreventive agent. Glutathione S‐transferase A1 (GSTA1) acts as a vital liver detoxification enzyme. Hepatocyte nuclear factor 1 (HNF‐1) regulates various cellular signaling pathways. In this study, we investigated the effects of C2‐ceramide and oltipraz on APAP‐induced hepatocyte injury and the changes of HNF‐1 and GSTA1. Results showed that C2‐ceramide (6 μmol/L) exacerbated APAP‐induced hepatocyte injury and caused a significant decrease (P < .01) in HNF‐1 and GSTA1 expressions. Meanwhile, GSTA1 content in supernatant was significantly increased (P < .01). In contrast, oltipraz (8 μmol/L) reduced the injury and significantly elevated (P < .01) HNF‐1 and GSTA1 expressions while GSTA1 content in supernatant was significantly decreased (P < .01). In conclusion, these findings revealed that C2‐ceramide inhibited HNF‐1 and GSTA1 expression and exacerbated hepatocyte injury, while oltipraz treatment results in the reduction of hepatocyte injury, and promoted HNF‐1 and GSTA1 expression. Additionally, the changes in HNF‐1 and GSTA1 were related to APAP‐induced hepatocyte injury. These results were useful to investigate the mechanism of an antipyretic and analgesic drug combination.