Structural and Functional Aspects of the Hepatic Sinusoids

Burt, Alastair David (1991) Structural and Functional Aspects of the Hepatic Sinusoids. MD thesis, University of Glasgow.

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The hepatic sinusoids were long regarded as simple, inert vascular channels lined by phagocytic and non-phagocytic cells derived from a common progenitor cell. This thesis describes studies dealing with various structural and functional aspects of the mammalian sinusoids. The aims of the work included can be broadly summarized as follows: (i) To investigate the role of sinusoidal cells in the synthesis of complement proteins. (ii) To characterize the composition of the extracellular matrix of the space of Disse in normal liver and to examine alterations to this in liver disease. (iii) To identify the cellular origin of such extracellular matrix proteins with particular attention to the role of sinusoidal cells. (iv) Having identified fat-storing cells as a major source of extracellular matrix proteins in (iii), to study the response of these cells in vivo to acute and chronic liver injury. (v) To identify and characterize intra-sinusoidal nerve fibres and to examine alterations to sinusoidal innervation in liver disease. The recently developed methods of sinusoidal cell isolation and culture were used to investigate the capacity of Kupffer cells, sinusoidal endothelial cells and fat-storing cells to synthesize C3, the most abundant protein of the complement cascade pathway. C3 was detected in Kupffer cell supernatants using a sensitive ELISA. Release of C3 could be reversibly inhibited by addition of the protein synthesis inhibitor cycloheximide, indicating active synthesis of the protein by these cells. Secretion of C3 could be enhanced by the prior exposure of the cells to bacterial endotoxin. Smaller amounts of C3 were also detected in fat-storing cell cultures after 5 days in culture but were undetectable in sinusoidal endothelial cell cultures. This study was the first to demonstrate C3 synthesis and secretion by sinusoidal cells. Although it was calculated that Kupffer cells are likely to contribute less than 1% of total hepatic C3 production, the observation of enhanced secretion following exposure to endotoxin suggests that C3 release by these cells may play an important role in the local intrahepatic host response to bacterial toxins. In order to characterize the extracellular matrix of the space of Disse in human liver, a panel of affinity-purified polyclonal antibodies to a wide range of matrix components was used for immunolocalization of the proteins by light microscopy and an ultracryomicrotomy/ immuno-gold labelling method was applied for their identification at the ultrastructural level in liver biopsy specimens. The normal space of Disse was found to contain several types of interstitial collagen (types I, III and V), a "minor" collagen (type VI), fibronectin, undulin and vitronectin. In spite of the paucity of a recognisable basement membrane at this site, the basement membrane components collagen type IV and laminin could also be identified. Increased immunolabelling for fibronectin was noted in the space of Disse in material from patients with acute liver disease. By contrast, in various forms of chronic (fibrotic) liver disease increased labelling was noted for interstitial collagens (types I and III) and the basement membrane proteins collagen type IV and laminin. Such changes in the extracellular matrix of the space of Disse may adversely affect parenchymal cell function by altering normal cell-matrix interactions and may perpetuate liver injury by interfering with the transport of nutrients between the sinusoidal blood flow and parenchymal cells. Two methods were used to examine the role of sinusoidal cells in the synthesis of extracellular matrix proteins. First, immunohistochemistry was used to identify intracellular proteins by light microscopy and at the ultrastructural level. Second, in vitro biosynthesis of matrix proteins by isolated rat liver sinusoidal cells was studied using [3H]-proline incorporation and SDS-PAGE analysis. Evidence is presented which suggests that fat-storing cells are a major source of such proteins; these cells may therefore be important in hepatic fibrogenesis. In order to study the response of these cells in vivo to various forms of experimental liver injury, a method for their immunolocalization in rat liver using monoclonal antibodies to the intermediate filament protein desmin was developed. This method was used to monitor the response of fat-storing cells to two forms of experimental injury: acute carbon tetrachloride-induced damage and chronic cholestatic injury (common bile duct ligation). In both models, desmin-positive fat-storing cells accumulated in areas of parenchymal cell damage. A novel double-labelling method was used to demonstrate that expansion of the desmin-positive fat-storing cell population was, at least in part, due to local cell proliferation. Furthermore, in both models evidence of phenotypic modulation in these cells towards that of myofibroblasts was found by demonstration of expression of the alpha-(smooth muscle) isoform of actin. (Abstract shortened by ProQuest.).

Item Type: Thesis (MD)
Qualification Level: Doctoral
Keywords: Medicine, Pathology
Date of Award: 1991
Depositing User: Enlighten Team
Unique ID: glathesis:1991-78326
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 28 Feb 2020 12:09
Last Modified: 28 Feb 2020 12:09

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