Changes In Sinusoidal Endothelial Cells Research Articles

МОРФОЛОГИЧЕСКИЕ ИЗМЕНЕНИЯ СОСУДИСТОЙ СИСТЕМЫ ПЕЧЕНИ КРЫС ПОД ВОЗДЕЙСТВИЕМ ТИОАЦЕТАМИДА

Background. Angiogenesis plays a key role in the progression of liver fibrosis. However, the available data on morphological changes in the liver vascular system are insufficient and contradictory. Objective. The aim of the work is to study the morphological changes in the liver vascular system of rats under the influence of thioacetamide. Material and methods. Fibrosis and cirrhosis of the liver in Wistar rats were induced with thioacetamide given at a dose of 200 mg/kg of animal weight for 17 weeks. To study morphological changes, we used classical and immunohistochemical staining methods. Microscopic analysis was performed using OLYMPUS BX51 microscope and image analysis software ImageScope Color and cellSens Standard. Results. The introduction of a solution of thioacetamide through the stomach leads to a gradual increase in the progression of pathological changes. In addition, it permits to track all stages of cirrhosis development and morphological restructuring of the liver vascular system. Throughout the experiment there was intensive capillarization of the parenchyma sinusoids and neoangiogenesis in the portal tracts and connective tissue septa manifested by the formation of many venules and small veins. We also observed an increase in the area of interlobular veins, which in some places had reached gigantic proportions. Three morphological phenotypes of CD34-positive cells were revealed. In the interlobular arteries as well as interlobular, central and sublobular veins, these cells had an elongated shape and a rod-shaped dark-colored nucleus. During the transformation of liver fibrosis into cirrhosis CD34-positive cells of an elongated shape with light roundedelongated nuclei were observed in the sinusoids closer to the periphery of individual false nodules. Rounded cells with dark-colored nuclei were present in the connective tissue near the hepatic triads, in the connective tissue septa among the cells of the infiltrate and between the sharply increased number of bile ducts. Conclusions. The established complex phenotypic changes in sinusoidal endothelial cells prove a close connection between fibrogenesis and neoangiogenesis. They probably play a leading role in the development of fibrosis and restructuring of the venous system of the portal vein.

Protective Effect of Ischemic Preconditioning against Liver Injury after Major Hepatectomy Using the Intermittent Pringle Maneuver in Swine

Objective: To investigate whether ischemic preconditioning (IP) protects the liver against ischemia-reperfusion injury (I/R-I) after major hepatectomy through intermittent hepatic pedicle clamping (IC) in a swine liver resection model. Background: Although many studies have reported a protective effect of IP against continuous hepatic ischemia, it has not been elucidated whether IP protects the liver against I/R-I after hepatectomy using IC. This is the first study to evaluate the effect of IP in a swine major hepatectomy model using IC. Methods: Pigs (n = 12) were divided into 2 groups (IP or non-IP). In the IP group, livers were subjected to IP (10 min ischemia and 10 min reperfusion) before liver resection using IC (15 min ischemia and 5 min reperfusion). A left hemihepatectomy was then performed using IC in both groups. Hemodynamic changes and plasma concentrations of aspartate aminotransferase, lactate dehydrogenase, lactic acid and hyaluronic acid were measured at 60, 120 and 180 min after hepatectomy. Apoptosis (TUNEL staining and electron microscopy), plasma tumor necrosis factor-α (TNF-α) and NO₂^–/NO₃^– were evaluated for 180 min after hepatectomy. Results: There were no significant differences in body weight, blood loss, resected liver weight, Pringle time or hemodynamic changes between the 2 groups. IP significantly reduced plasma aspartate aminotransferase levels for 180 min after hepatectomy (IP: 135.8 ± 13.5 vs. non-IP: 199 ± 16.8 IU/l; p = 0.018). In the non-IP group, apoptotic changes in sinusoidal endothelial cells were observed with increased plasma TNF-α levels. IP protected liver injury from increase in plasma TNF-α (p = 0.042). Significantly fewer apoptotic cells were seen in the IP than in the non-IP group (p = 0.002). Plasma levels of lactate dehydrogenase, lactic acid and NO₂^–/NO₃^– in the IP group tended to be lower than those in the non-IP group. Conclusions: IP prior to hepatectomy with IC resulted in less hepatic injury and apoptotic cell death than in livers not subjected to IP. IP with IC has the potential to improve the clinical postoperative course of patients undergoing hepatectomy.

Effect of S-nitrosoglutathione (GSNO) added to the University of Wisconsin solution (UW): II) functional response to cold preservation/reperfusion of rat liver

Livers cold preserved in University of Wisconsin (UW) solution followed by reperfusion suffer ischemia/reperfusion injuries. Microcirculation is the primary target of damage, characterized by sinusoidal perfusion failure due, mainly, to morphological changes of sinusoidal endothelial cells. Here, we demonstrated that the addition of S-nitrosoglutathione (GSNO) to the UW solution before cold storage, as a nitric oxide (NO) donor, attenuated hepatic injuries.Wistar adult rat livers were stored in UW solution (0°C – 48 hs) and then reperfused during 60 minutes using the Isolated Perfused Rat Model (IPRL). We assayed four GSNO concentration (50, 100, 250 and 500 μM). NO concentration was estimated calculating the amount of nitrite (NO2-) generated in the UW solution. Injuries during cold preservation were established measuring lactate dehydrogenase (LDH) released to the UW solution. Meanwhile, intrahepatic resistance (IR), LDH released to the perfusate, the effluent/perfusate ratio for K+, bile flow, liver glycogen content and sinusoidal endothelial cell morphology were studied after 1 hour of reperfusion in the IPRL system.In cold preserved livers without GSNO, glycogen content was dramatically reduced, IR increased markedly, LDH released was high, bile flow diminished and sinusoidal endothelial cells appeared rounded and detached from perisinusoidal matrix after reperfusion. The presence of 100 μM GSNO prevented the IR rise and LDH release, improved bile production and partially reduced endothelial cells damages.In conclusion, the addition of 100 μM GSNO to UW solution improved hemodynamic and function capacity of cold preserved/reperfused livers.

Role of sinusoidal endothelial cells in liver disease.

We characterized the structural changes of sinusoidal endothelial cells in chronic ethanol-fed rats and rats with cirrhosis induced by thioacetamide. The phenotypic changes of sinusoidal endothelial cells in fibrotic rats induced by thioacetamide and the reversibility of these changes were also investigated under transmission and scanning electron microscopy, regular microscopy and by immunohistochemistry with laminin and von Willebrand factor antibodies. The diameter and porosity of sinusoidal endothelial fenestrations were increased in chronic ethanol-fed rats without liver fibrosis, however, they decreased within 4 weeks of the cessation of thioacetamide treatment. A basement membrane-like structure in Disse's space was noted 6 weeks after thioacetamide treatment. Laminin was detected in Disse's space after 4 weeks and von Willebrand factor was detected in the cytoplasm as granular fluorescence after 6 weeks of thioacetamide treatment. Reversibility of the phenotypic changes of the sinusoidal endothelial cells was demonstrated in fibrotic liver of rats that received thioacetamide for 6 weeks after long-term discontinuation of thioacetamide administration. These results indicate that the structural and immunohistochemical characteristics of sinusoidal endothelial cells change in chronic ethanol-fed rats and fibrotic rats and these changes are reversible.

Sinusoidal capillarization in small hepatocellular carcinoma.

The morphological and phenotypic changes of sinusoidal endothelial cells in hepatocellular carcinoma were investigated along with the hemodynamic changes. Hepatocellular carcinomas, which were 10-20 mm in diameter, were obtained by liver aspiration biopsy. Twenty specimens of well differentiated and 20 specimens of moderately differentiated hepatocellular carcinoma were used. These were classified into two groups of hepatocellular carcinoma: with and without hypervascularity. Electron microscopy, immunohistochemistry using antibodies against factor VIII-related antigen, type IV collagen and laminin and lectin histochemistry using Ulex europaeus agglutinin I (UEA-I) were performed. Factor VIII-related antigen and UEA-1 binding sites were present in the sinusoidal endothelial cells in two groups. In hepatocellular carcinoma with hypervascularity, type IV collagen and laminin were present corresponding to basement membranes along the endothelial cells, which had few fenestrae. In another group, although type IV collagen was present along the endothelial cells that had a few fenestrae, laminin and basement membranes were rarely observed. These results suggest that the sinusoidal endothelial cells tend to show phenotypic changes in the early stage of hepatocarcinogenesis. As the arterial blood supply for hepatocellular carcinoma increases, the sinusoidal endothelial cells may form basement membranes and take on the morphological appearance of capillaries.

Defenestration of the sinusoidal endothelial cell in a rat model of cirrhosis

We characterized the structural and immunohistological changes of sinusoidal endothelial cells that occur during cirrhosis in rats made cirrhotic with thioacetamide. Thioacetamide (200 mg/kg body wt) was injected intraperitoneally three times a week into male Wistar rats. Two, 4, 6 and 12 wk later, rat livers were observed under transmission and scanning electron microscopy and regular microscopy and immunostained with laminin and von Willebrand factor (factor VIII-related antigen) antibodies. The diameters and numbers of sinusoidal endothelial fenestrations did not change significantly after 2 wk in the thioacetamide-treated rats; however, they decreased within 4 wk after thioacetamide treatment. A basement membranelike structure in Disse's space was noted 6 wk after thioacetamide treatment. Laminin was detected in Disse's space after 4 wk. In vitro, in cultured sinusoidal endothelial cells, the diameter of sinusoidal endothelial fenestrations was significantly lower at 6 wk in thioacetamide-treated rats. von Willebrand factor was detected in the cytoplasm as granular fluorescence after 6 wk of thioacetamide treatment. These results suggest that as fibrosis develops in cirrhosis, the structural and immunohistochemical characteristics of sinusoidal endothelial cells change.