Scott L. Friedman

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.

Molecular mechanisms of hepatic fibrosis and principles of therapy.

Tremendous insights into the understanding of hepatic fibrosis have taken place over the past ten years. Foremost among these is the recognition that hepatic stellate cells (formerly known as lipocytes, Ito cells, or fat-storing cells) play a central role based on their ability to undergo activation following liver injury of any cause. Stellate cell activation is a broad phenotypic response, characterized by distinct functional changes in proliferation, contractility, fibrogenesis, cytokine secretion, and matrix degradation. Insights gained into the molecular regulation of hepatic stellate cell activation will lead to new, targeted approaches to hepatic fibrosis in the future, and could lead to reduced morbidity and mortality in patients with chronic liver injury.

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.

Early genes induced in hepatic stellate cells during wound healing.

Activation of mesenchymal cells is a central event in the wound healing response of most tissues. In liver, the mesenchymal element responsible for organ fibrosis is the hepatic stellate cell (HSC) (formerly known as lipocyte or Ito cell). The phenotypic cascade of stellate cell activation in liver fibrosis has been well documented and involves both marked morphologic changes and upregulation of several functional components including extracellular matrix, cytokine receptors, contractile filaments and metalloproteinases. However, the genetic regulation of stellate cell activation is poorly understood. In an attempt to clone genes that are involved in the regulation of HSC activation we have combined cDNA library amplification by PCR with subtraction hybridization/differential screening, and have successfully identified genes induced in vivo during early stellate cell activation in a rat model of liver fibrosis. The subtracted cDNA library comprised less than 100 unique sequences. Of these, 13 clones with sizes ranging from 322 to 745 were sequenced and characterized. Gene induction in HSCs was monitored by RNAse protection assay during early liver injury induced by the hepatotoxin CCl4. The sequenced cDNAs corresponding to the known genes included type II transforming growth factor beta receptor, glutathione peroxidase I, transferrin and several clones encoding cellular retrotransposons, whose expression was not previously identified in non-parenchymal liver cells. In addition, one partial cDNA predicted a zinc-finger motif, suggesting a possible role of a novel transcriptional regulator. Our approach represents a valuable strategy for clarifying in vivo regulatory mechanisms of mesenchymal cell activation in wound healing.

Downregulation of matrix and β-PDGF receptor gene expression by anti-TGFβ antibody in rat hepatic stellate cells during experimental liver injury

Abstract Excess matrix in hepatic fibrosis results from both fibrogenic stimulation of stellate cells by TGFβ1 and cell proliferation due to induction of β-platelet derived growth factor receptor (β-PDGFR). In this paper, treatment of culture-activated rat stellate cells with anti-TGFβ inhibited collagen and fibronectin mRNA expression by 82 and 58%, respectively, versus control cells. In vivo, anti-TGFβ inhibited collagen I gene expression by 86% in stellate cells isolated from rats treated with CC14 compared with control antibody. In contrast to stellate cells, anti-TGFβ had no effect on collagen I gene expression in isolated sinusoidal endothelial cells. Anti-TGFβ administered in vivo to rats with liver injury also reduced expression of stellate cell β-PDGFR mRNA to that of control animals. Anti-TGFβ antibody had no effect on the histologic appearance of the tissue. These data support a role for TGFβ in stellate cell matrix expression and provide evidence for transmodulation of PDGF receptor by TGFβ in vivo. However, inhibition of TGFβ alone may not be adequate to attenuate severe hepatic injury and fibrosis.