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D M Bissell

Publications and source records attributed to D M Bissell.

At least 37 records · Page 2Linked to original sources
PubMed1993

Endothelin receptors in rat liver: lipocytes as a contractile target for endothelin 1.

The endothelins (ETs) form a group of three vasoactive peptides (ET-1, ET-2, and ET-3) for which two types of cellular receptors have been identified, types A and B ET receptors (ETA and ETB receptors, respectively). To address possible targets for ETs within the liver, we isolated the four principal liver cell populations and placed them in short-term primary culture. By ligand-binding assay and mRNA levels, expression of ET receptors was greatest on hepatic lipocytes (Ito cells or fat-storing cells), which are perisinusoidal cells exhibiting features of smooth muscle cells. Moreover, lipocytes expressed both ETA and ETB receptors. The mRNA for ETB receptor, but not for ETA receptor, was detectable in sinusoidal endothelial cells and Kupffer cells; neither mRNA was detectable in hepatocytes. Both ET-1 and ET-3 elicited contraction of activated lipocytes cultured on collagen lattices; the EC50 value for ET-1 was 3 +/- 1 nM and for ET-3 was 17 +/- 12 nM. In cell isolates from injured liver (after administration of carbon tetrachloride), expression of ET receptors was unchanged. However, mRNA for ET-1 was significantly increased in activated lipocytes, suggesting an autocrine loop for the initiation of lipocyte contraction. The findings imply that ET-1 may play a role in regulating sinusoidal perfusion through its effect on lipocytes, particularly in injury states.

Adipocytes↗
PubMed1993

Cell-matrix interactions in liver.

Studies of cell-matrix interaction in liver have demonstrated the biological impact of extracellular matrix on the structure and function of liver cells, both parenchymal and mesenchymal. Much of the work involves cell culture models, in which either hepatocytes or non-parenchymal liver cells are plated on various extracellular matrix proteins and the expression of tissue-specific function is assessed. The data suggest that a basement membrane-like matrix exists within the perisinusoidal space and is critical to the maintenance of normal liver function. There are reservations concerning the accuracy of cell-culture models with respect to the intact liver, in that the precise composition and structure of this matrix still is uncertain. Nonetheless, work to date has added a new dimension to the role of the extracellular matrix of the normal liver and a new appreciation of the potential impact of pathologically altered matrix ('fibrosis') in liver disease.

Amino Acid Sequence↗
PubMed1992

Cationic lipid-mediated transfection of liver cells in primary culture.

We describe transfection of DNA into parenchymal and individual non-parenchymal cell populations from adult rat liver in early primary culture, using cationic lipid as the carrier. All cell populations were transfectable, although lipid requirements varied by cell type and, for hepatocytes, with the age of the culture. For hepatocytes in early primary culture (2-10 hours after plating), pure DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) was strikingly more effective than commercial formulations (Lipofectin or TransfectACE) containing components in addition to, or other than DOTMA. For hepatocytes fully adapted to culture (approximately 48 hours after plating), pure DOTMA and Lipofectin were similarly effective. Under optimal conditions, about 10% of hepatocytes expressed the transfected reporter gene. CAT expression in hepatocytes doubled from 48 hours to 7 days after transfection. The effect of culture substratum on transfection efficiency also was examined. The presence of basement membrane-like matrix (EHS gel) reduced uptake of the DNA-lipid complex. However, cells in early culture that were transfected on collagen and then replated on EHS gel, displayed significantly greater reporter gene activity than did cells maintained throughout on collagen. In contrast to hepatocytes, non-parenchymal cells (lipocytes, Kupffer cells and endothelial cells, respectively) were transfected most efficiently by Lipofectin; DOTMA alone was inactive. The methods described will facilitate studies of gene regulation in individual liver cell populations.

Animals↗
PubMed1992

Role of extracellular matrix in regulating fenestrations of sinusoidal endothelial cells isolated from normal rat liver.

Open fenestrations are a conspicuous feature of sinusoidal endothelial cells and allow free movement of plasma into the space of Disse. In hepatic fibrosis, the number of fenestrations decreases as interstitial collagen increases in the liver, a change that correlates with deposition of extracellular matrix in the space of Disse. In this study, the possibility of a causal relationship between altered fenestral morphology and perisinusoidal matrix has been examined by culturing rat sinusoidal endothelial cells on individual matrix proteins or on a native matrix consisting of human amniotic membrane with interstitial collagen (types I and III) on one side and basement membrane proteins (collagen types IV and V and laminin) on the other. Under culture conditions, individual components of the extracellular matrix failed to maintain fenestrations. A basement-membranelike gel matrix derived from the Engelbreth-Holm-Swarm tumor war similarly ineffective. Fenestral density and porosity (percentage of cell surface occupied by fenestrations) were significantly enhanced, however, when endothelial cells were cultured on the basement-membrane side of human amnion. These data suggest that support of endothelial fenestrations requires a complex matrix. In particular, physiologically derived basement membrane maintains fenestrations, whereas interstitial collagen matrix does not. The loss of fenestrations associated with hepatic fibrosis may be related in part to an accumulation of interstitial collagens in the space of Disse.

Amnion↗
PubMed1991

Iron mediates production of a neutrophil chemoattractant by rat hepatocytes metabolizing ethanol.

Ethanol metabolism in hepatocytes is accompanied by release of a potent lipid chemoattractant for neutrophils. Production of the factor may initiate the inflammation associated with alcoholic hepatitis. In previous studies with a cytosol system from liver, production was blocked by iron chelators as well as by catalase and superoxide dismutase, suggesting the involvement of oxyradicals in formation of the chemoattractant. These studies have examined the role of iron in intact hepatocytes using cells from rats fed an iron-deficient diet, a control diet or a diet containing 3% carbonyl iron. The iron content averaged 1.4 nmol/mg protein in iron-deficient cells, 6.3 in controls and 135.3 in iron-loaded cells. Hepatocytes from all groups were established in primary culture and incubated with ethanol (10 mM); the medium was assayed for chemoattractant activity for human neutrophils. Cultures from chow-fed or iron-loaded animals produced chemoattractant as previously reported. By contrast, chemoattractant production was undetectable in the iron-deficient cultures. Addition of ferric citrate (10 microM) restored chemoattractant production while increasing cellular iron in the deficient cells less than 50% (to 2.3 nmol/mg protein). Addition of desferrioxamine mesylate to cultures of iron-loaded cells ablated chemoattractant production. The data provide evidence for the importance of hepatocellular iron in production of this alcohol-related lipid chemoattractant and suggest that a small intracellular pool of "free" iron plays a critical role.

Animals↗
PubMed1990

Hepatic fibrosis: new insights into pathogenesis.

From available clinical and experimental data, the deterioration of hepatocellular function associated with fibrosis may be ascribed, in part, to loss of biologic support provided by a normal extracellular matrix. These studies of pathogenesis are expected to clarify new points of therapeutic intervention and also to rationalize empiric therapies.

Basement Membrane↗
PubMed1990

Connective tissue biology and hepatic fibrosis: report of a conference.

The past 15 years have seen major advances in the characterization of extracellular matrix proteins and structure of matrix. As a by-product of this work, investigators now have an array of molecular and immunological reagents for monitoring matrix metabolism. Progress in the isolation and culture of individual cell types from liver has made possible direct measurement of matrix protein production and also has opened the way to studies of matrix degradation. The expanding knowledge of soluble mediators of inflammation is being applied to the regulation of matrix protein synthesis and degradation. Finally, experimental models of fibrosis in vivo are available for defining the complexity of matrix metabolism in the intact tissue and for validating the findings from cell culture and in vitro systems.

Animals↗
PubMed1990

Hepatic fibrosis: cellular aspects.

The space of Disse in the liver contains ECM proteins that have the function of - and may constitute- a low-density basement membrane. Specific functions of both hepatocytes and lipocytes appear to depend on the type of ECM in contact with these cells. The ECM of the space of Disse is altered in inflammation with deposition of ectopic proteins and degradation of normal constituents. Activation of lipocytes is a central event and responsible for the elaboration of an abnormal ECM; lipocytes also secrete a matrix proteinase. From the available clinical and experimental data, the deterioration of hepatocellular function associated with fibrosis may be ascribed, in part, to loss of the biologic support provided by a normal ECM.

Basement Membrane↗
PubMed1990

Transcriptional regulation of the albumin gene in cultured rat hepatocytes. Role of basement-membrane matrix.

The expression of tissue-specific functions by hepatocytes in primary culture is enhanced in the presence of an extracellular matrix. A basement membrane-like substratum, derived from the Engelbreth-Holm-Swarm mouse sarcoma (EHS) and termed EHS gel, supports synthesis and secretion of albumin for at least three weeks, in contrast to a conventional substratum (plastic or collagen-coated plastic), on which cells rapidly lose this function. The presence of an EHS matrix (as a substratum or added to the medium as a dilute gel) supports transcriptional activity at 30 to 35% and specific mRNA at 70 to 80% of initial values after five days of culture, at a time when transcription in cells plated in conventional culture is undetectable. For examining the cis elements required for transcriptional regulation by EHS matrix, we are utilizing recombinant adenoviruses to introduce DNA into hepatocytes, as an alternative to transfection of DNA fragments. Initial studies are presented, in which hepatocytes are cultured on either collagen-coated plastic or on EHS gel. At various times after plating, the cultures are infected with an adenovirus containing the proximal 5' regulatory region (to -441 base-pair) of the albumin gene. The results indicate no effect of EHS gel on this proximal promoter region, implying that matrix-responsive element(s) lie further upstream, possibly within the previously described enhancer at about -10,000 base-pairs.

Adenoviridae Infections↗
PubMed1989

Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix.

This study examined the role of extracellular matrix in regulating matrix phenotype of hepatic lipocytes, the major source of matrix in liver. Lipocytes (Ito, stellate, or fat-storing cells) were purified from normal rat liver and established in primary culture on either uncoated plastic, plastic coated with individual matrix proteins, or a "complete" gel matrix, a basement membrane-like matrix derived from the Engelbreth-Holm-Swarm (EHS) murine tumor. The ultrastructure of lipocytes cultured on the gel matrix resembled that of cells in normal liver, whereas lipocytes on plastic had dispersed nuclear chromatin and expanded rough endoplasmic reticulum, consistent with active proliferation and secretion. Lipocytes on the gel matrix exhibited no proliferative activity; cells maintained on plastic proliferated and produced type I collagen predominantly. Total collagen secretion by lipocytes on the gel matrix was 29% of that of cells on plastic, and consisted of type III collagen only. This difference extended to proteoglycan production, which was less than 5% of the amount produced by cells in conventional culture on plastic. The effects of the EHS gel were not reproduced by the individual components of the gel (laminin, type IV collagen, and heparan sulfate proteoglycan) or by a type I collagen gel. They were also reversible upon transfer of the cells to conventional culture. In contrast to lipocytes, collagen synthesis by hepatocytes was similar whether cultured on EHS gel or on plastic. These results show that the extracellular matrix can modulate matrix protein production by lipocytes and imply that, in early hepatic inflammation, changes in the hepatic subendothelial matrix may underlie stimulation of lipocyte matrix production and progression of the fibrotic process.

Animals↗
PubMed1989

Lipocytes from normal rat liver release a neutral metalloproteinase that degrades basement membrane (type IV) collagen.

We report a proteinase that degrades basement-membrane (type IV) collagen and is produced by the liver. Its cellular source is lipocytes (fat-storing or Ito cells). Lipocytes were isolated from normal rat liver and established in primary culture. The cells synthesize and secrete a neutral proteinase, which by gelatin-substrate gel electrophoresis and gel filtration chromatography, has a molecular mass of 65,000 D. The enzyme is secreted in latent form and is activated by p-aminophenylmercuric acetate but not by trypsin. Enzyme activity in the presence of EDTA is restored selectively by zinc and is unaffected by serine-protease inhibitors. In assays with radiolabeled soluble substrates, it degrades native type IV (basement membrane) collagen but not interstitial collagen types I or V and exhibits no activity against laminin or casein. At temperatures causing partial denaturation of soluble collagen in vitro, it rapidly degrades types I and V. Thus, it is both a type IV collagenase and gelatinase. The enzyme may play a role in initiating breakdown of the subendothelial matrix in the Disse space as well as augmenting the effects of collagenases that attack native interstitial collagen.

Animals↗
PubMed1988

Formation of extracellular matrix in normal rat liver: lipocytes as a major source of proteoglycan.

Proteoglycans are a major component of the normal hepatic extracellular matrix and undergo quantitative and qualitative changes in hepatic fibrosis. The cellular sources of proteoglycans are as yet incompletely defined. We examined this question using primary cultures of hepatocytes and lipocytes isolated from normal rat liver. Proteoglycan synthesis was assessed by measuring production of sulfated glycosaminoglycan, the polysaccharide moiety of proteoglycans. The findings indicate that lipocytes produce sixfold more glycosaminoglycan, per cell, than do hepatocytes. Two-thirds of the newly synthesized material is cell- or matrix-associated. Of the individual glycosaminoglycan species produced by lipocytes, dermatan sulfate represents 60% of the total; heparan sulfate and chondroitin sulfate are measurable but relatively minor. In hepatocyte cultures, heparan sulfate accounted for essentially all of the glycosaminoglycan detected. We conclude that lipocytes are an important source of proteoglycan in normal liver and may be the principal source of dermatan sulfate associated with hepatic fibrosis.

Animals↗
PubMed1988

Immunolocalization of laminin in normal rat liver and biosynthesis of laminin by hepatic lipocytes in primary culture.

Laminin, a glycoprotein with a molecular weight of approximately 850,000 daltons, is a major constituent of most epithelial basement membranes. Its presence in the extracellular matrix of normal liver, however, is debated. Using two affinity-purified antibodies directed against laminin, we have localized the glycoprotein within normal rat liver and identified its cellular source. Immunofluorescent staining of rat liver sections revealed laminin in a continuous distribution around hepatic sinusoids, adjacent to hepatocytes and sinusoidal lining cells. To determine the cellular origin of laminin, three perisinusoidal cell populations (hepatocytes, sinusoidal endothelial cells, and lipocytes) were purified from enzymatically dispersed rat liver and were established in primary culture. By immunofluorescence, laminin was associated almost exclusively with lipocytes. Synthesis of laminin was demonstrated by immunoprecipitation of the protein from lipocyte culture medium pulse-labeled with radioactive methionine. These results show that in adult liver, laminin is present in the perisinusoidal matrix and is produced by hepatic lipocytes. Lipocytes, which have the capacity to produce collagen as well as laminin, may be the principal source of extracellular matrix proteins in the perisinusoidal space, and may contribute to subendothelial fibrosis resulting from liver injury.

Animals↗
PubMed1988

Treatment of acute hepatic porphyria with hematin.

The efficacy of hematin has been evaluated in eight patients with acute intermittent porphyria: six with acute attacks and two with chronic subacute symptoms. Hematin suppressed the chemical signs of porphyria in all patients and the symptoms in those with acute attacks. The clinical response to hematin occurred uniformly on the third or fourth day of treatment, suggesting that a stereotypic response to this form of therapy can be defined. Hematin had no effect on chronic subacute symptoms. The indications for hematin therapy, its rationale and the details of its administration are reviewed.

Acute Disease↗
PubMed1988

Collagen measured in primary cultures of normal rat hepatocytes derives from lipocytes within the monolayer.

The cellular origin of hepatic collagen is under active investigation. Several recent studies using cells in primary culture suggest that hepatocytes are the source of much of the collagen in normal rat liver. In view of other data indicating that lipocytes produce substantial amounts of this protein, we have reexamined collagen biosynthesis in hepatocyte cultures that have been carefully characterized with respect to the presence of lipocytes. We find that routinely prepared hepatocyte isolates contain, by number, approximately 10% lipocytes. Lipocytes in early culture are difficult to visualize by phase-contrast microscopy but after 4 d proliferate and eventually replace the parenchymal cells. The size of the lipocyte subpopulation in these cultures correlates positively with collagen production. Similarly, removal of lipocytes by further processing of the initial hepatocyte isolate significantly reduces collagen production. Moreover, the only cells within hepatocyte cultures that display type I collagen by immunohistochemistry are lipocytes. We conclude that lipocytes are the principal source of collagen in primary hepatocyte cultures. The findings indicate also that these cells are the previously described "fibroblast" that appear in relatively long-term hepatocyte cultures.

Animals↗