directly by activating the matrix metalloproteinases (MMPs), Urokinase plasminogen activator (uPA) generates interstitial collagenase, and stromelysin. 4,5 Between them plasmin, a process inhibited by plasminogen-activator these enzymes are capable of degrading most matrix compoinhibitor (PAI)-1 and localized to the cell surface by nents. 6 Matrix remodeling is localized and directed by binding of uPA to a specific receptor. Plasmin degrades binding of uPA to a high-affinity cell surface receptor. 7 The extracellular matrix (ECM) both directly and by activaactivity of uPA is also modulated by specific plasminogention of matrix metalloproteinases (MMPs). Because stelactivator inhibitors (PAIs). Of these, PAI-1 is the most effilate cells play a central role in the pathogenesis of liver cient inhibitor in plasma. 8 fibrosis both via production of ECM proteins and Hepatic stellate cells (HSCs, also called Ito cells, fat-storing through secretion of MMPs, their contribution to plascells, or lipocytes) play a key role in matrix turnover in liver min generation was assessed. Stellate cells were preand are considered pivotal to the pathogenesis of liver fibropared from rat liver and cultured on plastic. Northern sis. 9 Stellate cells are situated in the space of Disse, have a analysis showed cellular expression of messenger RNA physiological role in retinoid storage, and contain cellular (mRNA) for PAI-1, uPA, and uPA receptor. Zymography/ retinoic acid-binding protein and nuclear retinoic acid-bindreverse zymography identified cell-surface-associated ing receptors. 10,11 In areas of liver injury, these cells prolifer-uPA activity and uPA and PAI-1 in culture media. Net ate and develop a myofibroblast-like phenotype. 12 Cellular uPA activity in culture media was maximal after 7 days transformation is accompanied by loss of retinoid and may in culture and then declined, whereas PAI-1 antigen levplay a causal role in HSC activation. 13,14 If plated on either els remained consistently elevated between 7 and 21 collagen I or plastic, HSCs undergo phenotypical and funcdays in culture. Stellate cell-mediated plasmin generational changes reminiscent of those observed in HSCs in tion was also seen in in vitro cultures supplemented with fibrosing liver. Cultured HSCs adopt a myofibroblast-like plasminogen. Because hepatic stellate cells (HSCs) conmorphology, express smooth muscle a-actin, and secrete coltain retinoids and release them on activation, the effect lagens (predominantly types I and III), 15 laminin, 16 proteoglyof retinoic acid on the plasminogen-activating system cans, 17 and fibronectin. 18 As well as being the major source was also assessed. Treatment of cultured HSCs with retiof matrix components in the liver, recent evidence shows that noic acid (1 mmol/L) increased uPA secretion 2.6-fold but HSCs participate in matrix remodeling through the synthesis did not alter PAI-1. We conclude that stellate cells synof the matrix metalloproteinases interstitial collagenase, 19 thesize key components of the plasminogen-activating stromelysin, 20 and gelatinase A. 21 They also secrete the major system and generate plasmin and therefore have the inhibitor of metalloproteinases, TIMP-1. 22 ability to regulate MMP activation. Upregulation of uPA
In this study we show that HSCs may further regulate synthesis by retinoic acid may have implications in mamatrix turnover by synthesizing components of the plasminotrix remodeling in sites of stellate cell activation in gen-activating system. Because HSCs contain retinoids and which high concentrations of retinoids may be achieved.
release them on activation, the effect of exogenous retinoic (HEPATOLOGY 1996;24:1172-1178.)
acid on the plasminogen-activating system was also assessed. The significance of these findings to the regulation of matrix Urokinase plasminogen activator (uPA) lies at the top of a degradation in the liver is discussed. proteolytic cascade shown to control matrix remodeling in a wide range of conditions including ovulation, embryogenesis,
Methods
angiogenesis, and tumor metastasis. 1,2 Urokinase acts to gen-Stellate Cell Isolation. Stellate cells were isolated from normal rat erate plasmin from circulating plasminogen. Plasmin is a liver (male Sprague-Dawley rats, 450-600 g). All animals received broad-spectrum proteinase capable of directly degrading mahumane care in compliance with British Home Office guidelines. trix components, including fibronectin, laminin, and proteo-Stellate cells were dispersed from liver by a combination of pronase glycans. 3 Plasmin also participates in matrix degradation inand collagenase perfusion and purified by Nycodenz density gradient centrifugation and centrifugal elutriation as previously described. 20 Stellate cells were plated at a density of 2 1 10 6 cells/well in uncoated plastic 35-mm dishes (for activity measurements) and divided slide Abbreviations: uPA, urokinase plasminogen activator; MMP, matrix metalloproteinase; wells for immunohistochemistry, or at a density of 6.5 1 10 6 in 25-PAI, plasminogen-activator inhibitor; HSC, hepatic stellate cell; DMEM, Dulbecco's modimm 2 flasks (for RNA extraction) in Dulbecco's modified Eagle mefied Eagle medium; FCS, fetal calf serum; cDNA, complementary DNA; DIG, digoxygenin; dium (DMEM; Imperial, Andover, England) supplemented with 20% PBS, phosphate-buffered saline; SSC, standard saline citrate; SDS, sodium dodecyl sulfate;
fetal calf serum (FCS), penicillin (10 mU/mL), streptomycin (10 mg/ mRNA, messenger RNA; UTP, uridine triphosphate; EDTA, ethylenediaminetetraacetic mL; Imperial), and gentamicin (32 mg/mL; Roussel, Dublin, Ireland). acid; TBS, Tris-buffered saline; Ig, immunoglobulin; ELISA, enzyme-linked immunosorbent assay; CBC, carbonate-bicarbonate coating; TGF-b1, transforming growth factor b1.