The role of hepatic stellate cells in liver regeneration

Abstract

Liver regeneration is an extremely complex process. Cell types and mechanisms involved depend on the extent of liver damage (mild to severe), the type of damage (with or without necrosis, inflammation), the underlying liver disease (acute or chronic), and the capacity of the whole body to respond (i.e. age). In clinical practice, regeneration is of prime importance in living organ transplantation, fulminant hepatitis and cirrhosis.The liver is normally proliferatively quiescent, but hepatocyte loss through partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response from all cell types in the liver to perfectly restore liver mass [[1]Alison M.R Vig P Russo F Bigger B.W Amofah E Themis M et al.Hepatic stem cells: from inside and outside the liver?.Cell Prolif. 2004; 37: 1-21Crossref PubMed Scopus (140) Google Scholar]. Hepatocytes are themselves the functional stem cells of the liver. More severe liver injury can activate a potential stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of bipotential transit amplifying cells (oval cells), that can ultimately differentiate into hepatocytes and biliary epithelial cells. One third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells may contribute to the albeit low renewal rate of hepatocytes, but can make a more significant contribution to regeneration under a very strong positive selection pressure. In such instances, cell fusion rather than transdifferentiation appears to be the underlying mechanism by which the haematopoietic genome becomes reprogrammed.This unique objective of the liver to reconstitute its liver mass and organization is even seen in culture [[2]Michalopoulos G.K Bowen W.C Mule K Stolz D.B Histological organization in hepatocyte organoid cultures.Am J Pathol. 2001; 159: 1877-1887Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar]. Hepatocytes isolated by collagenase perfusion of the liver and maintained in defined culture conditions undergo a series of complex changes, including apoptosis and cell proliferation, to reconstruct tissue with specific architecture. Cultures in collagen-coated pleated surface roller bottles, with hepatocyte growth medium and in the presence of hepatocyte growth factor (HGF) and epidermal growth factor (EGF), form characteristic and reproducible tissue architecture composed of a superficial layer of biliary epithelial cells, an intermediate layer of connective tissue and hepatocytes, and a basal layer of endothelial cells. Dexamethasone, EGF, and HGF are required for the complete histological organization. This reconstitution is possible because isolated hepatocytes contain contaminants such as hepatic stellate cells (HSCs) and probably other cell types such as sinusoidal endothelial cells (SECs).The HSCs are closely associated with SEC in the like manner of the pericytes to the capillaries. They are organized into a sheath surrounding the sinusoid network. In the normal human and rat liver, a basal lamina-like substance is interposed between the two cell types [3Wake K Structure of the sinusoidal wall in the liver.Cell Hepat. Sinusoid. 1995; : 241-246Google Scholar, 4Pattanapen G Ekataksin W The porcine arachnocytes spectrum: panzonal polymorphism of hepatic stellate cell population as revealed by extensive reconstruction of confocal optical imaging.Hepatology. 2003; 38 (abstract): 784ACrossref Google Scholar, 5Burt A.D Le Bail B Balabaud C Bioulac-Sage P Morphologic investigation of sinusoidal cells.Semin Liver Dis. 1993; 13: 21-38Crossref PubMed Scopus (53) Google Scholar].The subendothelial processes of the HSC terminate as very thin thorn-like microprojections. These microprojections, also seen on the cytoplasmic processes of cultured HSCs, are unique and represent one of the most characteristic structures of the HSCs. They make contacts with the plasma membrane of the hepatic parenchymal cells (HPCs). Thus the HSCs adhere to the SECs and also to the HPCs with these numerous hairs [3Wake K Structure of the sinusoidal wall in the liver.Cell Hepat. Sinusoid. 1995; : 241-246Google Scholar, 4Pattanapen G Ekataksin W The porcine arachnocytes spectrum: panzonal polymorphism of hepatic stellate cell population as revealed by extensive reconstruction of confocal optical imaging.Hepatology. 2003; 38 (abstract): 784ACrossref Google Scholar, 5Burt A.D Le Bail B Balabaud C Bioulac-Sage P Morphologic investigation of sinusoidal cells.Semin Liver Dis. 1993; 13: 21-38Crossref PubMed Scopus (53) Google Scholar].HSCs (also called arachnocytes) varied in shape from the periphery to the center of lobules. Morphological observations imply that the so-called activation of HSCs is facilitated if no contacts with sinusoid or hepatic plate are established [[3]Wake K Structure of the sinusoidal wall in the liver.Cell Hepat. Sinusoid. 1995; : 241-246Google Scholar].Hepatic regeneration consists of orchestrated multistep mechanisms that prime quiescent HPCs to undergo replication, allowing the liver to concomitantly grow, and terminating cell proliferation once the liver reaches the appropriate mass [6Vogten J.M Smakman N Voest E.E Borel Rinkes I.H Intravital analysis of microcirculation in the regenerating mouse liver.J Surg Res. 2003; 113: 264-269Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 7Mabuchi A Mullaney I Sheard P.W Hessian P.A Mallard B.L Tawadrous M.N et al.Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat.J Hepatol. 2004; 40: 910-916Abstract Full Text Full Text PDF PubMed Google Scholar, 8Wack K.E Ross M.A Zegarra V Sysko L.R Watkins S.C Stolz D.B Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver.Hepatology. 2001; 33: 363-378Crossref PubMed Scopus (141) Google Scholar]. Many growth factors and cytokines play important roles in each step. Tumor necrosis factor (TNF) and interleukin 6 have been implicated for the initial priming process. Once HPCs enter a state of replicative competence, they can fully respond to hepatocyte growth factor and transforming growth factor (TGF)-α. Thereafter, at the late phase, the growth process is negatively controlled by TGF-β and activin and terminates when it reaches a set point.Few data are available on the activation state and morphology of the HSCs of regenerating liver [6Vogten J.M Smakman N Voest E.E Borel Rinkes I.H Intravital analysis of microcirculation in the regenerating mouse liver.J Surg Res. 2003; 113: 264-269Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 7Mabuchi A Mullaney I Sheard P.W Hessian P.A Mallard B.L Tawadrous M.N et al.Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat.J Hepatol. 2004; 40: 910-916Abstract Full Text Full Text PDF PubMed Google Scholar, 8Wack K.E Ross M.A Zegarra V Sysko L.R Watkins S.C Stolz D.B Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver.Hepatology. 2001; 33: 363-378Crossref PubMed Scopus (141) Google Scholar]. The involvement of HSCs in liver regeneration is nevertheless widely accepted. They do proliferate, peaking at 48 h post-PHx, losing much of their lipid in the process. As a result, the HSC/SEC ratio is the greatest at 72 h post-PHx, allowing for the greatest interaction between these two cell types at a critical period of neovascularization during regeneration following PHx. It is likely that such a ratio could facilitate the vascularization process via close exchange of growth, matrix, and chemotactic factors.Surprisingly, very little is known about the ultrastructural changes at the sinusoidal surface during the regeneration and revascularization events that accompany compensatory hyperplasia. A consistent finding in regenerating liver is the loss of the typical plate-like architecture with formation of hepatocyte-clusters containing 8–10 cells without intervening sinusoids resulting in loss of the unique hepatocyte–vascular relationship. Mabuchi et al. [[7]Mabuchi A Mullaney I Sheard P.W Hessian P.A Mallard B.L Tawadrous M.N et al.Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat.J Hepatol. 2004; 40: 910-916Abstract Full Text Full Text PDF PubMed Google Scholar] have confirmed using intravital fluorescence microscopy, the presence of hepatocyte clusters at both 3 and 7 days after PHx. By day 3 after PHx, the presence of these hepatocyte-clusters interrupted the sinusoid thereby shortening its overall length. In addition, the average width of hepatic cords was increased significantly. By day 7 after PHx, the length of the sinusoid was again normal. The fact that functional vessel surface area remains normal until day 14 post PHx indicates compensatory vascular growth mechanisms to ensure adequate hepatocyte perfusion during liver regeneration. Why avascular hepatocyte-clusters form could relate to the different rate of proliferation of hepatocytes and SECs whose peak mitotic activities occur at ∼24 h and 4 days, respectively. Thus, rapid hepatocyte proliferation leads to the development of hepatocyte-clusters. Thereafter SECs proliferate and migrate into the hepatocyte-clusters and restore the normal sinusoidal architecture, although hepatocyte-clusters were still found in the liver after 7 days in both control and regenerating liver. At day 3 after PHx, the distance between HSCs falls to ∼36 μm and in consequence the HSC/hepatocyte ratio rose by ∼50%. In addition, HSCs congregated around the avascular hepatocyte islands indicating that direct HSC–HSC and/or HSC–hepatocyte interaction may occur in the regenerating liver. Immunohistochemistry revealed the presence of HSC-clusters particularly after 3 days and on occasions HSCs had already invaded hepatocyte-clusters in the early stages of regeneration (by day 1 and 3 after PHx). Many of the HSCs in the regenerating liver were activated (α-smooth muscle actin (SMA)-expressing).Similarly, HSC-clusters frequently contained α-SMA-expressing HSCs and were in close association with hepatocytes indicating the possibility that activation of HSCs and HSC–hepatocyte interaction were related events during regeneration.ECM degradation occurs in the sinusoids immediately following PHx. This degradation of ECM correlates with the up-regulation of urokinase plasminogen activator enzymatic activity and increased expression of its receptor in the liver within 1 min after PHx. Reduction in sinusoidal fibrinogen expression within the sinusoid remained until at least 24 h post-PHx. Such ECM degradation may be responsible for fenestration dilation. Several ECM components within the space of Disse undergo modulation throughout the regenerative process. While collagen I and III deposition do not appear to change dramatically, collagen IV, fibronectin, and laminin, have been shown to display differential deposition within sinusoids at various times. The plasmin/α2-antiplasmin (AP) system is critical to the maintenance of hemostasis and vascular potency through the degradation of fibrin, and seems to be important to a variety of physiological processes. Plasminogen (Plg)−/− mice showed abnormal liver regeneration with fibrin deposits after PHx. The livers of Plg−/− and Plg−/−.α2-AP−/− mice remained in the damaged state until 14 days after carbon tetrachloride (CCl4) injection. The injection of anti-α2-AP antibody in wt mice improved the regeneration after the liver injury, and the injection of tranexamic acid in α2-AP−/− mice reduced it. These results suggest that the plasmin/α2-AP system regulates HSC-induced production and the protease-induced degradation of fibronectin after toxic injury in mice [[9]Okada K Ueshima S Imano M Kataoka K Matsuo O The regulation of liver regeneration by the plasmin/alpha 2-antiplasmin system.J Hepatol. 2004; 40: 110-116Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar].After a single injection of high-dose dimethylnitrosamine (DMN), no HSC survived in the submassive hemorrhagic necrotic areas. Afterwards, HSCs in the preserved liver parenchyma were activated, proliferated, and migrated into the necrotic areas. Subsequent replacement of necrotic areas by regenerating hepatocytes and fibrosis may indicate that the activated HSCs play a significant role not only in the remodeling of sinusoidal wall necessary for the regeneration of hepatocytes but also in the formation of central–central bridging fibrosis at the site of the incomplete regeneration [[10]Jin Y.L Enzan H Kuroda N Hayashi Y Nakayama H Zhang Y.H et al.Tissue remodeling following submassive hemorrhagic necrosis in rat livers induced by an intraperitoneal injection of dimethylnitrosamine.Virchows Arch. 2003; 442: 39-47PubMed Google Scholar].The use of transgenic models has yielded information on how abnormal function of HSCs translates into regenerative defects. Foxf1 (a transcription factor) is expressed in HSCs of the developing and adult liver. In response to CCl4 injury, Foxf1+/− mice exhibited defective HSCs activation with a significant reduction in type I collagen and α-SMA protein levels. This was associated with an abnormal liver repair despite normal cellular proliferation, likely due to increased apoptosis. These results suggest that the defect in HSCs activation consecutive to haploinsufficiency of Foxf1 results in impaired regeneration [[11]Kalinichenko V.V Bhattacharyya D Zhou Y Gusarova G.A Kim W Shin B et al.Foxf1+/− mice exhibit defective stellate cell activation and abnormal liver regeneration following CCl4 injury.Hepatology. 2003; 37: 107-117Crossref PubMed Scopus (118) Google Scholar].Col-1α1r/rmice harbor mutations around the single collagenase cleavage site in the α1(I) chain of type I collagen that renders type I collagen completely resistant to digestion by all collagenolytic matrix metalloproteinases tested so far. Following CCl4 injury, these mice show persistent activation of HSCs and fail to regenerate properly. In the context of progressive fibrosis, this inhibition of hepatocyte proliferation may represent a significant mechanism preventing the restoration of effective hepatocellular function [[12]Issa R Zhou X Trim N Millward-Sadler H Krane S Benyon C et al.Mutation in collagen-1 that confers resistance to the action of collagenase results in failure of recovery from CCl4-induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration.FASEB J. 2003; 17: 47-49Crossref PubMed Scopus (174) Google Scholar].These two examples suggest that deficient as well as uncontrolled HSCs activation impairs liver regeneration. Thus, a finely tuned HSCs response may be an important factor to ensure adequate regeneration.Any new models to understand the regenerative process is welcome. In the present issue of J Hepatol, Mabuchi et al. have hypothesized that if HSC–HSC or hepatocyte–HSC cell adhesion takes place during early hepatic regeneration, the adhesions might survive cell isolation procedures. During the isolation of HPCs from normal liver, contamination by HSCs is usually <3%. Here they found that at PHx1 and PHx3, >20% of cells in HPC-fraction were HSCs. In addition, these HSCs were often isolated as cell clusters which mostly contained HSCs but also contained hepatocytes. Furthermore, they found that these HSCs were predominantly activated while HSCs in HSC-fraction were quiescent. By day 7 after PHx, HSCs are no longer found in the HPC-fraction. Thus, in the early stages of regeneration, HSCs form contacts with other HSCs and hepatocytes which then precipitate together during cell isolation. Their findings support the exciting possibility that hepatocyte–HSC interaction in the early stages after PHx (1–3 days) is intimately involved in the regeneration process.This study raises several questions:The origin and type of HSC involved. Clearly, more investigation is required to elucidate the identity, mechanism, and activation state of the HSC under PHx and characterize its role in the revascularization process. Presently we do not know exactly to what extent HSCs are truly in an activated state and which cell subtypes are involved and even if these cells are HSCs or other fibrogenic liver cells [[13]Ramadori G Saile B Portal tract fibrogenesis in the liver.Lab Invest. 2004; 84: 153-159Crossref PubMed Scopus (176) Google Scholar]. Indeed, whereas portal myofibroblasts could be subcultured several times, activated HSC from rat liver do not proliferate but become polyploid by endoreplication and died by apoptosis. This crucial difference allowed to discover differences in the gene-expression profiles between liver fibrogenic cell types (portal tract (myo)fibroblasts, and fibroblasts, including the second layer cells of the central veins and the fibroblasts of the liver capsule and HSCs). These data suggest that in the fibrogenic process myofibroblasts may migrate from the portal tract into the developing septa.The nature of the contact between hepatocytes and hepatic stellate cells. The cellular and molecular mechanisms underlying the activation of hepatocytes and the activation trigger have been investigated intensively; however, cell–cell adhesion function in the regenerating liver has received scant attention. The cellular mechanisms underlying hepatocyte/HSC adhesion and HSCs activation in the regenerating liver are currently unknown. Changes that occur in hepatocytes during proliferation may stimulate hepatocyte interaction with HSCs possibly via cell adhesion molecules or other factors leading to HSCs activation and HSCs clustering. These activated HSCs could then participate in the restoration of normal sinusoidal structure. HSCs group around SECs in the area adjacent to hepatocyte clusters in regenerating rat liver. It is plausible that HSCs and SECs accumulate near avascular hepatocyte clusters to facilitate development of new sinusoid channels through the newly proliferated hepatocyte.Impaired hepatic regeneration is a major problem after surgical resection of tumors from cirrhotic patients. It was shown that lipopolysaccharide (LPS) injected intraperitoneally to mice 24 h prior a 67% hepatectomy impaired liver regeneration. The following mechanism was unravelled [[14]Akita K Okuno M Enya M Imai S Moriwaki H Kawada N et al.Impaired liver regeneration in mice by lipopolysaccharide via TNF-alpha/kallikrein-mediated activation of latent TGF-beta.Gastroenterology. 2002; 123: 352-364Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. LPS stimulates Kupffer cells to secrete TNF-α. TNF-α enhances cell surface plasmakallicrein activity in HSCs. Plasma kallicrein provokes the proteolytic activation of latent TGF-β on the HSC surface. The resultant active TGF-β suppresses proliferation of HPCs, leading to impaired liver regeneration.It is through the deciphering of cross talks between the different liver cell types directly and through the ECM (both varying according to pathological models and time) that we shall be able to control liver regeneration in liver disease. Liver regeneration is an extremely complex process. Cell types and mechanisms involved depend on the extent of liver damage (mild to severe), the type of damage (with or without necrosis, inflammation), the underlying liver disease (acute or chronic), and the capacity of the whole body to respond (i.e. age). In clinical practice, regeneration is of prime importance in living organ transplantation, fulminant hepatitis and cirrhosis. The liver is normally proliferatively quiescent, but hepatocyte loss through partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response from all cell types in the liver to perfectly restore liver mass [[1]Alison M.R Vig P Russo F Bigger B.W Amofah E Themis M et al.Hepatic stem cells: from inside and outside the liver?.Cell Prolif. 2004; 37: 1-21Crossref PubMed Scopus (140) Google Scholar]. Hepatocytes are themselves the functional stem cells of the liver. More severe liver injury can activate a potential stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of bipotential transit amplifying cells (oval cells), that can ultimately differentiate into hepatocytes and biliary epithelial cells. One third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells may contribute to the albeit low renewal rate of hepatocytes, but can make a more significant contribution to regeneration under a very strong positive selection pressure. In such instances, cell fusion rather than transdifferentiation appears to be the underlying mechanism by which the haematopoietic genome becomes reprogrammed. This unique objective of the liver to reconstitute its liver mass and organization is even seen in culture [[2]Michalopoulos G.K Bowen W.C Mule K Stolz D.B Histological organization in hepatocyte organoid cultures.Am J Pathol. 2001; 159: 1877-1887Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar]. Hepatocytes isolated by collagenase perfusion of the liver and maintained in defined culture conditions undergo a series of complex changes, including apoptosis and cell proliferation, to reconstruct tissue with specific architecture. Cultures in collagen-coated pleated surface roller bottles, with hepatocyte growth medium and in the presence of hepatocyte growth factor (HGF) and epidermal growth factor (EGF), form characteristic and reproducible tissue architecture composed of a superficial layer of biliary epithelial cells, an intermediate layer of connective tissue and hepatocytes, and a basal layer of endothelial cells. Dexamethasone, EGF, and HGF are required for the complete histological organization. This reconstitution is possible because isolated hepatocytes contain contaminants such as hepatic stellate cells (HSCs) and probably other cell types such as sinusoidal endothelial cells (SECs). The HSCs are closely associated with SEC in the like manner of the pericytes to the capillaries. They are organized into a sheath surrounding the sinusoid network. In the normal human and rat liver, a basal lamina-like substance is interposed between the two cell types [3Wake K Structure of the sinusoidal wall in the liver.Cell Hepat. Sinusoid. 1995; : 241-246Google Scholar, 4Pattanapen G Ekataksin W The porcine arachnocytes spectrum: panzonal polymorphism of hepatic stellate cell population as revealed by extensive reconstruction of confocal optical imaging.Hepatology. 2003; 38 (abstract): 784ACrossref Google Scholar, 5Burt A.D Le Bail B Balabaud C Bioulac-Sage P Morphologic investigation of sinusoidal cells.Semin Liver Dis. 1993; 13: 21-38Crossref PubMed Scopus (53) Google Scholar]. The subendothelial processes of the HSC terminate as very thin thorn-like microprojections. These microprojections, also seen on the cytoplasmic processes of cultured HSCs, are unique and represent one of the most characteristic structures of the HSCs. They make contacts with the plasma membrane of the hepatic parenchymal cells (HPCs). Thus the HSCs adhere to the SECs and also to the HPCs with these numerous hairs [3Wake K Structure of the sinusoidal wall in the liver.Cell Hepat. Sinusoid. 1995; : 241-246Google Scholar, 4Pattanapen G Ekataksin W The porcine arachnocytes spectrum: panzonal polymorphism of hepatic stellate cell population as revealed by extensive reconstruction of confocal optical imaging.Hepatology. 2003; 38 (abstract): 784ACrossref Google Scholar, 5Burt A.D Le Bail B Balabaud C Bioulac-Sage P Morphologic investigation of sinusoidal cells.Semin Liver Dis. 1993; 13: 21-38Crossref PubMed Scopus (53) Google Scholar]. HSCs (also called arachnocytes) varied in shape from the periphery to the center of lobules. Morphological observations imply that the so-called activation of HSCs is facilitated if no contacts with sinusoid or hepatic plate are established [[3]Wake K Structure of the sinusoidal wall in the liver.Cell Hepat. Sinusoid. 1995; : 241-246Google Scholar]. Hepatic regeneration consists of orchestrated multistep mechanisms that prime quiescent HPCs to undergo replication, allowing the liver to concomitantly grow, and terminating cell proliferation once the liver reaches the appropriate mass [6Vogten J.M Smakman N Voest E.E Borel Rinkes I.H Intravital analysis of microcirculation in the regenerating mouse liver.J Surg Res. 2003; 113: 264-269Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 7Mabuchi A Mullaney I Sheard P.W Hessian P.A Mallard B.L Tawadrous M.N et al.Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat.J Hepatol. 2004; 40: 910-916Abstract Full Text Full Text PDF PubMed Google Scholar, 8Wack K.E Ross M.A Zegarra V Sysko L.R Watkins S.C Stolz D.B Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver.Hepatology. 2001; 33: 363-378Crossref PubMed Scopus (141) Google Scholar]. Many growth factors and cytokines play important roles in each step. Tumor necrosis factor (TNF) and interleukin 6 have been implicated for the initial priming process. Once HPCs enter a state of replicative competence, they can fully respond to hepatocyte growth factor and transforming growth factor (TGF)-α. Thereafter, at the late phase, the growth process is negatively controlled by TGF-β and activin and terminates when it reaches a set point. Few data are available on the activation state and morphology of the HSCs of regenerating liver [6Vogten J.M Smakman N Voest E.E Borel Rinkes I.H Intravital analysis of microcirculation in the regenerating mouse liver.J Surg Res. 2003; 113: 264-269Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 7Mabuchi A Mullaney I Sheard P.W Hessian P.A Mallard B.L Tawadrous M.N et al.Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat.J Hepatol. 2004; 40: 910-916Abstract Full Text Full Text PDF PubMed Google Scholar, 8Wack K.E Ross M.A Zegarra V Sysko L.R Watkins S.C Stolz D.B Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver.Hepatology. 2001; 33: 363-378Crossref PubMed Scopus (141) Google Scholar]. The involvement of HSCs in liver regeneration is nevertheless widely accepted. They do proliferate, peaking at 48 h post-PHx, losing much of their lipid in the process. As a result, the HSC/SEC ratio is the greatest at 72 h post-PHx, allowing for the greatest interaction between these two cell types at a critical period of neovascularization during regeneration following PHx. It is likely that such a ratio could facilitate the vascularization process via close exchange of growth, matrix, and chemotactic factors. Surprisingly, very little is known about the ultrastructural changes at the sinusoidal surface during the regeneration and revascularization events that accompany compensatory hyperplasia. A consistent finding in regenerating liver is the loss of the typical plate-like architecture with formation of hepatocyte-clusters containing 8–10 cells without intervening sinusoids resulting in loss of the unique hepatocyte–vascular relationship. Mabuchi et al. [[7]Mabuchi A Mullaney I Sheard P.W Hessian P.A Mallard B.L Tawadrous M.N et al.Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat.J Hepatol. 2004; 40: 910-916Abstract Full Text Full Text PDF PubMed Google Scholar] have confirmed using intravital fluorescence microscopy, the presence of hepatocyte clusters at both 3 and 7 days after PHx. By day 3 after PHx, the presence of these hepatocyte-clusters interrupted the sinusoid thereby shortening its overall length. In addition, the average width of hepatic cords was increased significantly. By day 7 after PHx, the length of the sinusoid was again normal. The fact that functional vessel surface area remains normal until day 14 post PHx indicates compensatory vascular growth mechanisms to ensure adequate hepatocyte perfusion during liver regeneration. Why avascular hepatocyte-clusters form could relate to the different rate of proliferation of hepatocytes and SECs whose peak mitotic activities occur at ∼24 h and 4 days, respectively. Thus, rapid hepatocyte proliferation leads to the development of hepatocyte-clusters. Thereafter SECs proliferate and migrate into the hepatocyte-clusters and restore the normal sinusoidal architecture, although hepatocyte-clusters were still found in the liver after 7 days in both control and regenerating liver. At day 3 after PHx, the distance between HSCs falls to ∼36 μm and in consequence the HSC/hepatocyte ratio rose by ∼50%. In addition, HSCs congregated around the avascular hepatocyte islands indicating that direct HSC–HSC and/or HSC–hepatocyte interaction may occur in the regenerating liver. Immunohistochemistry revealed the presence of HSC-clusters particularly after 3 days and on occasions HSCs had already invaded hepatocyte-clusters in the early stages of regeneration (by day 1 and 3 after PHx). Many of the HSCs in the regenerating liver were activated (α-smooth muscle actin (SMA)-expressing). Similarly, HSC-clusters frequently contained α-SMA-expressing HSCs and were in close association with hepatocytes indicating the possibility that activation of HSCs and HSC–hepatocyte interaction were related events during regeneration. ECM degradation occurs in the sinusoids immediately following PHx. This degradation of ECM correlates with the up-regulation of urokinase plasminogen activator enzymatic activity and increased expression of its receptor in the liver within 1 min after PHx. Reduction in sinusoidal fibrinogen expression within the sinusoid remained until at least 24 h post-PHx. Such ECM degradation may be responsible for fenestration dilation. Several ECM components within the space of Disse undergo modulation throughout the regenerative process. While collagen I and III deposition do not appear to change dramatically, collagen IV, fibronectin, and laminin, have been shown to display differential deposition within sinusoids at various times. The plasmin/α2-antiplasmin (AP) system is critical to the maintenance of hemostasis and vascular potency through the degradation of fibrin, and seems to be important to a variety of physiological processes. Plasminogen (Plg)−/− mice showed abnormal liver regeneration with fibrin deposits after PHx. The livers of Plg−/− and Plg−/−.α2-AP−/− mice remained in the damaged state until 14 days after carbon tetrachloride (CCl4) injection. The injection of anti-α2-AP antibody in wt mice improved the regeneration after the liver injury, and the injection of tranexamic acid in α2-AP−/− mice reduced it. These results suggest that the plasmin/α2-AP system regulates HSC-induced production and the protease-induced degradation of fibronectin after toxic injury in mice [[9]Okada K Ueshima S Imano M Kataoka K Matsuo O The regulation of liver regeneration by the plasmin/alpha 2-antiplasmin system.J Hepatol. 2004; 40: 110-116Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar]. After a single injection of high-dose dimethylnitrosamine (DMN), no HSC survived in the submassive hemorrhagic necrotic areas. Afterwards, HSCs in the preserved liver parenchyma were activated, proliferated, and migrated into the necrotic areas. Subsequent replacement of necrotic areas by regenerating hepatocytes and fibrosis may indicate that the activated HSCs play a significant role not only in the remodeling of sinusoidal wall necessary for the regeneration of hepatocytes but also in the formation of central–central bridging fibrosis at the site of the incomplete regeneration [[10]Jin Y.L Enzan H Kuroda N Hayashi Y Nakayama H Zhang Y.H et al.Tissue remodeling following submassive hemorrhagic necrosis in rat livers induced by an intraperitoneal injection of dimethylnitrosamine.Virchows Arch. 2003; 442: 39-47PubMed Google Scholar]. The use of transgenic models has yielded information on how abnormal function of HSCs translates into regenerative defects. Foxf1 (a transcription factor) is expressed in HSCs of the developing and adult liver. In response to CCl4 injury, Foxf1+/− mice exhibited defective HSCs activation with a significant reduction in type I collagen and α-SMA protein levels. This was associated with an abnormal liver repair despite normal cellular proliferation, likely due to increased apoptosis. These results suggest that the defect in HSCs activation consecutive to haploinsufficiency of Foxf1 results in impaired regeneration [[11]Kalinichenko V.V Bhattacharyya D Zhou Y Gusarova G.A Kim W Shin B et al.Foxf1+/− mice exhibit defective stellate cell activation and abnormal liver regeneration following CCl4 injury.Hepatology. 2003; 37: 107-117Crossref PubMed Scopus (118) Google Scholar]. Col-1α1r/rmice harbor mutations around the single collagenase cleavage site in the α1(I) chain of type I collagen that renders type I collagen completely resistant to digestion by all collagenolytic matrix metalloproteinases tested so far. Following CCl4 injury, these mice show persistent activation of HSCs and fail to regenerate properly. In the context of progressive fibrosis, this inhibition of hepatocyte proliferation may represent a significant mechanism preventing the restoration of effective hepatocellular function [[12]Issa R Zhou X Trim N Millward-Sadler H Krane S Benyon C et al.Mutation in collagen-1 that confers resistance to the action of collagenase results in failure of recovery from CCl4-induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration.FASEB J. 2003; 17: 47-49Crossref PubMed Scopus (174) Google Scholar]. These two examples suggest that deficient as well as uncontrolled HSCs activation impairs liver regeneration. Thus, a finely tuned HSCs response may be an important factor to ensure adequate regeneration. Any new models to understand the regenerative process is welcome. In the present issue of J Hepatol, Mabuchi et al. have hypothesized that if HSC–HSC or hepatocyte–HSC cell adhesion takes place during early hepatic regeneration, the adhesions might survive cell isolation procedures. During the isolation of HPCs from normal liver, contamination by HSCs is usually <3%. Here they found that at PHx1 and PHx3, >20% of cells in HPC-fraction were HSCs. In addition, these HSCs were often isolated as cell clusters which mostly contained HSCs but also contained hepatocytes. Furthermore, they found that these HSCs were predominantly activated while HSCs in HSC-fraction were quiescent. By day 7 after PHx, HSCs are no longer found in the HPC-fraction. Thus, in the early stages of regeneration, HSCs form contacts with other HSCs and hepatocytes which then precipitate together during cell isolation. Their findings support the exciting possibility that hepatocyte–HSC interaction in the early stages after PHx (1–3 days) is intimately involved in the regeneration process. This study raises several questions: The origin and type of HSC involved. Clearly, more investigation is required to elucidate the identity, mechanism, and activation state of the HSC under PHx and characterize its role in the revascularization process. Presently we do not know exactly to what extent HSCs are truly in an activated state and which cell subtypes are involved and even if these cells are HSCs or other fibrogenic liver cells [[13]Ramadori G Saile B Portal tract fibrogenesis in the liver.Lab Invest. 2004; 84: 153-159Crossref PubMed Scopus (176) Google Scholar]. Indeed, whereas portal myofibroblasts could be subcultured several times, activated HSC from rat liver do not proliferate but become polyploid by endoreplication and died by apoptosis. This crucial difference allowed to discover differences in the gene-expression profiles between liver fibrogenic cell types (portal tract (myo)fibroblasts, and fibroblasts, including the second layer cells of the central veins and the fibroblasts of the liver capsule and HSCs). These data suggest that in the fibrogenic process myofibroblasts may migrate from the portal tract into the developing septa. The nature of the contact between hepatocytes and hepatic stellate cells. The cellular and molecular mechanisms underlying the activation of hepatocytes and the activation trigger have been investigated intensively; however, cell–cell adhesion function in the regenerating liver has received scant attention. The cellular mechanisms underlying hepatocyte/HSC adhesion and HSCs activation in the regenerating liver are currently unknown. Changes that occur in hepatocytes during proliferation may stimulate hepatocyte interaction with HSCs possibly via cell adhesion molecules or other factors leading to HSCs activation and HSCs clustering. These activated HSCs could then participate in the restoration of normal sinusoidal structure. HSCs group around SECs in the area adjacent to hepatocyte clusters in regenerating rat liver. It is plausible that HSCs and SECs accumulate near avascular hepatocyte clusters to facilitate development of new sinusoid channels through the newly proliferated hepatocyte. Impaired hepatic regeneration is a major problem after surgical resection of tumors from cirrhotic patients. It was shown that lipopolysaccharide (LPS) injected intraperitoneally to mice 24 h prior a 67% hepatectomy impaired liver regeneration. The following mechanism was unravelled [[14]Akita K Okuno M Enya M Imai S Moriwaki H Kawada N et al.Impaired liver regeneration in mice by lipopolysaccharide via TNF-alpha/kallikrein-mediated activation of latent TGF-beta.Gastroenterology. 2002; 123: 352-364Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. LPS stimulates Kupffer cells to secrete TNF-α. TNF-α enhances cell surface plasmakallicrein activity in HSCs. Plasma kallicrein provokes the proteolytic activation of latent TGF-β on the HSC surface. The resultant active TGF-β suppresses proliferation of HPCs, leading to impaired liver regeneration. It is through the deciphering of cross talks between the different liver cell types directly and through the ECM (both varying according to pathological models and time) that we shall be able to control liver regeneration in liver disease.