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Desmin and actin as tools to monitor ito cells transformation to myofibroblasts in experimental liver fibrosis
WP-C5
IMMUNOGOLD LOCALIZATION OF PROCOLLAGEN TYPE I I I ON THE ULTRATHIN FROZENSECTIONS OF RAT AND HUMANLIVER. REINVESTIGATION OF THE RETICULAR FIBER CONCEPT
A. Geerts, D. Schuppan*, E. Hahn*, P. Van Der Seek**, P. Schellinck, R.B. De Zanier, E. Wisse. Lab voor Celbiologie en Histologie, Vrije Universiteit Brussel (V.U.B.), Belgie. *Universit~tsklinikum Steglitz, Freie Universit~t Berlin, BRD. **Akademisch Ziekenhuis, Vrije Universiteit Brussel, Belgie. Despite numerous studies on the occurrence and distribution of i n t e r s t i t i a l collagens in human and animal livers, i t is s t i l l uncertain whether collagen type I l l is exclusively present in fine collagen bundles, often described as reticular fibers, or codistributed with type I collagen in all i n t e r s t i t i a l collagen bundles present in the space of Disse. In the present study we have analyzed this problem in two ways : (i) procollagen type I l l (PIIIP) has been localized on ultrathin frozen sections of rat and human l i v e r by the protein A-gold technique, using a f f i n i t y purified a n t i - r a t procollagen type I l l antibodies and monospecific antihuman procollagen type I l l antiserum, and ( i i ) by measuring the collagen f i b r i l diameters and counting the numbers of f i b r i l s per collagen bundle. In both human and rat l i v e r , anti PIIIP antibodies react with a l l i n t e r s t i t i a l collagen f i b r i l s present in the space of Disse. No obvious difference in staining intensity was seen when solitary f i b r i l s were compared with f i b r i l s organized in bundles. Measurement of collagen f i b r i l diameters showed a unimodal distribution of the f i b r i l diameters with an average of 62.4 nm (SE = 0.16 nm) in the rat and of 57.3 nm (SE = 0.13 in man. In the rat l i v e r 90 % of the f i b r i l s present in the space of Disse, are organized in bundles containing less than 30 fibrils. From these results, i t is concluded that the classical "reticular fiber" concept in which i t is assumed that collagen type I l l is predominantly localized in small diameter f i b r i l s or bundles is not very l i k e l y to hold for the l i v e r .
WP-C6
OESMIN AND ACTIN AS TOOLS TO MONITOR ITO CELLS TRANSFORMATION TO IN EXPERIMENTAL LIVER FIBROSIS
MYOFIBROBLASTS
G_:_. 8allardini, A_=. Faccani, M_= Fallani, S_=_.8erti, *V. Vasi, *G~_.8ia~ini, F.B. Bianchi, E. Pisi. Clinica Medica II ed *Istologia. Universita' di Bologna. Ito cells (IC) and myofibroblasts (M) are easily identified on plastic embedded material on the basis of ultrastructural morphology and the presence of cytoplasmic lipid droplets. It has been reported Lhat M contain actin (A) and that both IC and M contain desmin (0}. Aim of this study was to evaluate the pattern of distribution of 0 (using a monoclonal antibody, 0E8-5, Boerhinger) and of A (using TRITC-conJugated ketophalloidin, which specifically binds to A) in an experimental model of liver fibrosis induced in rats by intraperitoneal injections oT heterologous (swine) serum. A double IFL tecnique was applied on cryostat liver sections from rats at different sLages of treatment. In normal liver O was found in smooth muscle cells of blood vessels, scanty fibroblasL-like cells in portal tracts and single sinusoidal cells (IC). Strong positivity for A was detected in vessel walls, the luminal portion of bile ducts and bile canaliculi; a faint staining was diffusely observed along sinusoids. In trea ted non fibrotic rats (5-10 injections) a clearcut increase in number and staining of lobular IC (D+, A-) was observed. The overall A pattern was unmodified. In fibrotic livers with low collagen content, large, highly cellular sepia (10-20 injections) contained a large number of both strongly O+, A- (IC) and O+,A+ (M) cells. Lobular IC (0+, A-) were normal in number and positivity. In advanced stages (20-40 injections) with high liver collagen content, thin, nearly acellular, septa displayed few 0+, A+ cells. These observations are in line with previous morphometric and ultrastructural observations and confirm that both IC and M are positive for O, while only M contain A. The modulation of IC to M is associated with increased 0 content and the appearence of A. Visualization of actin and desmin allows to monitor, with a relatively simple tecnique, proliferation of Ito cells, their accumulation in the septa and modulation to myofibroblasts and to indirectly evaluate ongoing fibrogenesis.
$51
IMMUNOGOLD LOCALIZATION OF PROCOLLAGEN TYPE I I I ON THE ULTRATHIN FROZENSECTIONS OF RAT AND HUMANLIVER. REINVESTIGATION OF THE RETICULAR FIBER CONCEPT
A. Geerts, D. Schuppan*, E. Hahn*, P. Van Der Seek**, P. Schellinck, R.B. De Zanier, E. Wisse. Lab voor Celbiologie en Histologie, Vrije Universiteit Brussel (V.U.B.), Belgie. *Universit~tsklinikum Steglitz, Freie Universit~t Berlin, BRD. **Akademisch Ziekenhuis, Vrije Universiteit Brussel, Belgie. Despite numerous studies on the occurrence and distribution of i n t e r s t i t i a l collagens in human and animal livers, i t is s t i l l uncertain whether collagen type I l l is exclusively present in fine collagen bundles, often described as reticular fibers, or codistributed with type I collagen in all i n t e r s t i t i a l collagen bundles present in the space of Disse. In the present study we have analyzed this problem in two ways : (i) procollagen type I l l (PIIIP) has been localized on ultrathin frozen sections of rat and human l i v e r by the protein A-gold technique, using a f f i n i t y purified a n t i - r a t procollagen type I l l antibodies and monospecific antihuman procollagen type I l l antiserum, and ( i i ) by measuring the collagen f i b r i l diameters and counting the numbers of f i b r i l s per collagen bundle. In both human and rat l i v e r , anti PIIIP antibodies react with a l l i n t e r s t i t i a l collagen f i b r i l s present in the space of Disse. No obvious difference in staining intensity was seen when solitary f i b r i l s were compared with f i b r i l s organized in bundles. Measurement of collagen f i b r i l diameters showed a unimodal distribution of the f i b r i l diameters with an average of 62.4 nm (SE = 0.16 nm) in the rat and of 57.3 nm (SE = 0.13 in man. In the rat l i v e r 90 % of the f i b r i l s present in the space of Disse, are organized in bundles containing less than 30 fibrils. From these results, i t is concluded that the classical "reticular fiber" concept in which i t is assumed that collagen type I l l is predominantly localized in small diameter f i b r i l s or bundles is not very l i k e l y to hold for the l i v e r .
WP-C6
OESMIN AND ACTIN AS TOOLS TO MONITOR ITO CELLS TRANSFORMATION TO IN EXPERIMENTAL LIVER FIBROSIS
MYOFIBROBLASTS
G_:_. 8allardini, A_=. Faccani, M_= Fallani, S_=_.8erti, *V. Vasi, *G~_.8ia~ini, F.B. Bianchi, E. Pisi. Clinica Medica II ed *Istologia. Universita' di Bologna. Ito cells (IC) and myofibroblasts (M) are easily identified on plastic embedded material on the basis of ultrastructural morphology and the presence of cytoplasmic lipid droplets. It has been reported Lhat M contain actin (A) and that both IC and M contain desmin (0}. Aim of this study was to evaluate the pattern of distribution of 0 (using a monoclonal antibody, 0E8-5, Boerhinger) and of A (using TRITC-conJugated ketophalloidin, which specifically binds to A) in an experimental model of liver fibrosis induced in rats by intraperitoneal injections oT heterologous (swine) serum. A double IFL tecnique was applied on cryostat liver sections from rats at different sLages of treatment. In normal liver O was found in smooth muscle cells of blood vessels, scanty fibroblasL-like cells in portal tracts and single sinusoidal cells (IC). Strong positivity for A was detected in vessel walls, the luminal portion of bile ducts and bile canaliculi; a faint staining was diffusely observed along sinusoids. In trea ted non fibrotic rats (5-10 injections) a clearcut increase in number and staining of lobular IC (D+, A-) was observed. The overall A pattern was unmodified. In fibrotic livers with low collagen content, large, highly cellular sepia (10-20 injections) contained a large number of both strongly O+, A- (IC) and O+,A+ (M) cells. Lobular IC (0+, A-) were normal in number and positivity. In advanced stages (20-40 injections) with high liver collagen content, thin, nearly acellular, septa displayed few 0+, A+ cells. These observations are in line with previous morphometric and ultrastructural observations and confirm that both IC and M are positive for O, while only M contain A. The modulation of IC to M is associated with increased 0 content and the appearence of A. Visualization of actin and desmin allows to monitor, with a relatively simple tecnique, proliferation of Ito cells, their accumulation in the septa and modulation to myofibroblasts and to indirectly evaluate ongoing fibrogenesis.
$51