Focus
Authors: Scott L. Friedman
Journal: Journal of Hepatology (2012)

A silent partner no longer – Sinusoidal endothelial cells in liver homeostasis and diseaseA study in this month’s issue of the Journal illustrates the growing appreciation for how non-parenchymal cells – in this case liver sinusoidal endothelial cells – contribute to disease. The term ‘non-parenchymal cells’ refers not only to endothelial cells, but also to hepatic stellate cells macrophages and a host of resident lymphocytes and immune cells within the liver, which collectively comprise approximately 40% of the total number of liver cells. Stellate cells have garnered most of our attention in the past decade because of their major contribution to fibrogenesis, and because of ready methods to isolate and characterize the cells. Moreover, stellate cells love to grow in culture and thus even mixed non-parenchymal cell cultures invariably become dominated by stellate cells when plated in the presence of serum. Macrophages, which in the liver have been traditionally called “Kupffer cells” based on the late 19th century description by Kupffer [[1]Wake K. Decker K. Kirn A. Knook D.L. McCuskey R.S. Bouwens L. et al.Cell biology and kinetics of Kupffer cells in the liver.Int Rev Cytol. 1989; 118: 173-229Crossref PubMed Scopus (150) Google Scholar], are also well appreciated. Moreover, we have adopted an increasingly nuanced understanding of their heterogeneity and divergent functions as a result of the identification of many cell surface markers, and advances in flow cytometry methods [[2]Duffield J.S. Dendritic cells take on more tasks in the liver?.Hepatology. 2012; 55: 16-19Crossref PubMed Scopus (2) Google Scholar].In contrast, the sinusoidal endothelial cell has been largely overlooked, in part because its wispy presence in tissue sections is easily disregarded, and also because the cell hates primary culture and is exquisitely sensitive to the stress of tissue disruption and isolation. Nonetheless, an enlarging biology has emerged, focused for the most part on their unique intracellular pores, or fenestrae, and their contribution to portal hypertension [[3]Iwakiri Y. Endothelial dysfunction in the regulation of cirrhosis and portal hypertension.Liver Int. 2012; 32: 199-213Crossref PubMed Scopus (128) Google Scholar]. ‘Capillarization’ of sinusoids with loss of these fenestrae in liver disease has been recognized for almost 50 years [[4]Schaffner F. Popper H. Capillarization of hepatic sinusoids in man.Gastroenterology. 1963; 44: 239-242Abstract Full Text PDF PubMed Scopus (503) Google Scholar], but the functional consequences of defenestration and its impact on sinusoidal homeostasis are only now being clarified. The cells are highly responsive to vascular mediators including vascular endothelial growth factor (VEGF), nitric oxide (NO), endothelin, thromboxanes, and prostaglandins that contribute to a dynamic crosstalk with hepatic stellate cells in regulating blood flow [[3]Iwakiri Y. Endothelial dysfunction in the regulation of cirrhosis and portal hypertension.Liver Int. 2012; 32: 199-213Crossref PubMed Scopus (128) Google Scholar]. In fact, maintenance of sinusoidal fenestrations and prevention of capillarization in vivo help preserve stellate cell quiescence and minimize fibrosis [[5]Xie G, Wang X, Wang L, Atkinson RD, Kanel GC, Gaarde WA, et al. Role of differentiation of liver sinusoidal endothelial cells in progression and regression of hepatic fibrosis in rats. Gastroenterology, in press. [Epub ahead of print].Google Scholar]. Conversely, liver injury provokes changes in sinusoidal endothelial and stellate cells that amplify each other’s pathogenic responses, leading to an accelerating pattern of increasing resistance to blood flow and accumulating fibrosis.The paper by Drs. Rodríguez-Villarupla and colleagues in this issue of the Journal builds on this model of paracrine regulation and introduces a new intracellular mediator of sinusoidal cell responses – peroxisome proliferator activated receptor alpha (PPARα). This molecule is perhaps the best-characterized among members of the nuclear hormone superfamily, and its classical function is to increase fatty acid oxidation by combining with the RXR nuclear receptor to stimulate lipid metabolism-regulating genes [[6]Pyper S.R. Viswakarma N. Yu S. Reddy J.K. PPARalpha: energy combustion, hypolipidemia, inflammation and cancer.Nucl Recept Signal. 2010; 8: e002Crossref PubMed Google Scholar]. Ligands for PPARα fall into two main groups – synthetic xenobiotics (exogenous) and biological molecules (endogenous). The best-known synthetic ligands are the ‘fibrates’ (e.g. clofibrate, fenofibrate), which are typically prescribed as hypolipidemic agents, whereas the endogenous ligands are primarily fatty acids and their derivatives. It is not surprising given its role in lipid metabolism that most previous studies examining PPARα biology in the liver have focused on hepatocytes. Yet, more recent studies have identified effects of PPARα in improving endothelial dysfunction in cardiac and vascular tissues [7Hamblin M. Chang L. Fan Y. Zhang J. Chen Y.E. PPARs and the cardiovascular system.Antioxid Redox Signal. 2009; 11: 1415-1452Crossref PubMed Scopus (159) Google Scholar, 8Hiukka A. Maranghi M. Matikainen N. Taskinen M.R. PPARalpha: an emerging therapeutic target in diabetic microvascular damage.Nat Rev Endocrinol. 2010; 6: 454-463Crossref PubMed Scopus (89) Google Scholar], and have uncovered PPARα target genes that regulate blood flow (e.g. thromboxanes) to account for some of these effects.In this context, it was logical to explore the potential contribution of PPARα activity to hepatic blood flow regulation in the Rodríguez-Villarupla study. Mice in which hepatic fibrosis was established by 3 weeks of CCl4 administration were then treated with either a control diet or one containing a pharmacologic concentration of fenofibrate for an additional week, and the extent of hepatic fibrosis and impact on hemodynamics were assessed. Remarkably, PPARα activation led to reduced hepatic fibrosis and portal pressure despite only a week of therapy, which was associated with improved vasodilatory responses to acetylcholine and increased bioavailability of nitric oxide (a vaso-relaxing compound) in endothelial cells from treated mice.These findings are very appealing because fibrates are already widely prescribed to patients with hyperlipidemia, and therefore their safety is established. Human trials would seem like a logical next step. However, the results really represent an initial series of observations that merit further exploration first. Specifically, the cellular targets of PPARα actions are not clarified, as the effects could represent solely the direct impact of fenofibrate on endothelial cells, or more likely the added effects of the drug on PPARα activity in hepatocytes and/or macrophages, which also express PPARα, but probably not on hepatic stellate cells, since prior studies indicate that they are not responsive to PPARα ligands [9Everett L. Galli A. Crabb D. The role of hepatic peroxisome proliferator-activated receptors (PPARs) in health and disease.Liver. 2000; 20: 191-199Crossref PubMed Scopus (109) Google Scholar, 10Miyahara T. Schrum L. Rippe R. Xiong S. Yee Jr., H.F. Motomura K. et al.Peroxisome proliferator-activated receptors and hepatic stellate cell activation.J Biol Chem. 2000; 275: 35715-35722Crossref PubMed Scopus (426) Google Scholar]. How, then, does PPARα activation reduce fibrosis? Based on the recent study by Xie et al. [[5]Xie G, Wang X, Wang L, Atkinson RD, Kanel GC, Gaarde WA, et al. Role of differentiation of liver sinusoidal endothelial cells in progression and regression of hepatic fibrosis in rats. Gastroenterology, in press. [Epub ahead of print].Google Scholar], an attractive hypothesis is that restoration of normal endothelial responses (i.e. reversal of capillarization) drives quiescence of stellate cells through an unknown mechanism. This would imply that in doing so, net protease activity is increased to help resorb accumulated scar and reduce fibrogenic gene expression. These effects of PPARα are likely to be indirect through induction of PPARα-regulated genes, either in endothelial cells, Kupffer cells and/or hepatocytes. The most elegant approach to sort out these possibilities would be to generate mice with cell-specific knockout of PPARα. Cell-specific depletion could be accomplished by using a Cre-lox strategy in which mice expressing Cre recombinase driven by a cell type-specific promoter (e.g. albumin-Cre for hepatocytes), could be crossed with animals in which the PPARα allele is flanked by LoxP sites (i.e. ‘floxed’). This would lead in the progeny to Cre-mediated recombination that deletes PPARα only in cells in which the promoter that is driving Cre expression is active. Such a strategy would enable investigators to determine in which cell type(s) fenofibrate is exerting its actions not only in regulating vasoregulation, which appears to be a direct effect on sinusoidal endothelium, but also in fibrosis, in which the effect on stellate cells is likely to be indirect.Based on the Rodríguez-Villarupla study and related papers, it is clear that the sinusoidal endothelial cell represents a fertile focus for more studies whose findings could directly improve treatments for liver disease. Although culture and characterization methods have been more challenging for this cell type, new tools are emerging to overcome these obstacles, including an immortalized murine sinusoidal endothelial cell line [[11]Huebert R.C. Jagavelu K. Liebl A.F. Huang B.Q. Splinter P.L. LaRusso N.F. et al.Immortalized liver endothelial cells: a cell culture model for studies of motility and angiogenesis.Lab Invest. 2010; 90: 1770-1781Crossref PubMed Scopus (46) Google Scholar], identification of new cell surface markers, and characterization of endothelial cell-specific promoters [[12]Ding B.S. Nolan D.J. Butler J.M. James D. Babazadeh A.O. Rosenwaks Z. et al.Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration.Nature. 2010; 468: 310-315Crossref PubMed Scopus (547) Google Scholar]. In addition to their role in vasoregulation, sinusoidal endothelium are also a driving force behind angiogenesis in liver development, regeneration, and cancer [[13]Fernandez M. Semela D. Bruix J. Colle I. Pinzani M. Bosch J. Angiogenesis in liver disease.J Hepatol. 2009; 50: 604-620Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar]. They should no longer be viewed as a silent partner in liver homeostasis.Conflict of interestThe author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. A silent partner no longer – Sinusoidal endothelial cells in liver homeostasis and disease A study in this month’s issue of the Journal illustrates the growing appreciation for how non-parenchymal cells – in this case liver sinusoidal endothelial cells – contribute to disease. The term ‘non-parenchymal cells’ refers not only to endothelial cells, but also to hepatic stellate cells macrophages and a host of resident lymphocytes and immune cells within the liver, which collectively comprise approximately 40% of the total number of liver cells. Stellate cells have garnered most of our attention in the past decade because of their major contribution to fibrogenesis, and because of ready methods to isolate and characterize the cells. Moreover, stellate cells love to grow in culture and thus even mixed non-parenchymal cell cultures invariably become dominated by stellate cells when plated in the presence of serum. Macrophages, which in the liver have been traditionally called “Kupffer cells” based on the late 19th century description by Kupffer [[1]Wake K. Decker K. Kirn A. Knook D.L. McCuskey R.S. Bouwens L. et al.Cell biology and kinetics of Kupffer cells in the liver.Int Rev Cytol. 1989; 118: 173-229Crossref PubMed Scopus (150) Google Scholar], are also well appreciated. Moreover, we have adopted an increasingly nuanced understanding of their heterogeneity and divergent functions as a result of the identification of many cell surface markers, and advances in flow cytometry methods [[2]Duffield J.S. Dendritic cells take on more tasks in the liver?.Hepatology. 2012; 55: 16-19Crossref PubMed Scopus (2) Google Scholar]. In contrast, the sinusoidal endothelial cell has been largely overlooked, in part because its wispy presence in tissue sections is easily disregarded, and also because the cell hates primary culture and is exquisitely sensitive to the stress of tissue disruption and isolation. Nonetheless, an enlarging biology has emerged, focused for the most part on their unique intracellular pores, or fenestrae, and their contribution to portal hypertension [[3]Iwakiri Y. Endothelial dysfunction in the regulation of cirrhosis and portal hypertension.Liver Int. 2012; 32: 199-213Crossref PubMed Scopus (128) Google Scholar]. ‘Capillarization’ of sinusoids with loss of these fenestrae in liver disease has been recognized for almost 50 years [[4]Schaffner F. Popper H. Capillarization of hepatic sinusoids in man.Gastroenterology. 1963; 44: 239-242Abstract Full Text PDF PubMed Scopus (503) Google Scholar], but the functional consequences of defenestration and its impact on sinusoidal homeostasis are only now being clarified. The cells are highly responsive to vascular mediators including vascular endothelial growth factor (VEGF), nitric oxide (NO), endothelin, thromboxanes, and prostaglandins that contribute to a dynamic crosstalk with hepatic stellate cells in regulating blood flow [[3]Iwakiri Y. Endothelial dysfunction in the regulation of cirrhosis and portal hypertension.Liver Int. 2012; 32: 199-213Crossref PubMed Scopus (128) Google Scholar]. In fact, maintenance of sinusoidal fenestrations and prevention of capillarization in vivo help preserve stellate cell quiescence and minimize fibrosis [[5]Xie G, Wang X, Wang L, Atkinson RD, Kanel GC, Gaarde WA, et al. Role of differentiation of liver sinusoidal endothelial cells in progression and regression of hepatic fibrosis in rats. Gastroenterology, in press. [Epub ahead of print].Google Scholar]. Conversely, liver injury provokes changes in sinusoidal endothelial and stellate cells that amplify each other’s pathogenic responses, leading to an accelerating pattern of increasing resistance to blood flow and accumulating fibrosis. The paper by Drs. Rodríguez-Villarupla and colleagues in this issue of the Journal builds on this model of paracrine regulation and introduces a new intracellular mediator of sinusoidal cell responses – peroxisome proliferator activated receptor alpha (PPARα). This molecule is perhaps the best-characterized among members of the nuclear hormone superfamily, and its classical function is to increase fatty acid oxidation by combining with the RXR nuclear receptor to stimulate lipid metabolism-regulating genes [[6]Pyper S.R. Viswakarma N. Yu S. Reddy J.K. PPARalpha: energy combustion, hypolipidemia, inflammation and cancer.Nucl Recept Signal. 2010; 8: e002Crossref PubMed Google Scholar]. Ligands for PPARα fall into two main groups – synthetic xenobiotics (exogenous) and biological molecules (endogenous). The best-known synthetic ligands are the ‘fibrates’ (e.g. clofibrate, fenofibrate), which are typically prescribed as hypolipidemic agents, whereas the endogenous ligands are primarily fatty acids and their derivatives. It is not surprising given its role in lipid metabolism that most previous studies examining PPARα biology in the liver have focused on hepatocytes. Yet, more recent studies have identified effects of PPARα in improving endothelial dysfunction in cardiac and vascular tissues [7Hamblin M. Chang L. Fan Y. Zhang J. Chen Y.E. PPARs and the cardiovascular system.Antioxid Redox Signal. 2009; 11: 1415-1452Crossref PubMed Scopus (159) Google Scholar, 8Hiukka A. Maranghi M. Matikainen N. Taskinen M.R. PPARalpha: an emerging therapeutic target in diabetic microvascular damage.Nat Rev Endocrinol. 2010; 6: 454-463Crossref PubMed Scopus (89) Google Scholar], and have uncovered PPARα target genes that regulate blood flow (e.g. thromboxanes) to account for some of these effects. In this context, it was logical to explore the potential contribution of PPARα activity to hepatic blood flow regulation in the Rodríguez-Villarupla study. Mice in which hepatic fibrosis was established by 3 weeks of CCl4 administration were then treated with either a control diet or one containing a pharmacologic concentration of fenofibrate for an additional week, and the extent of hepatic fibrosis and impact on hemodynamics were assessed. Remarkably, PPARα activation led to reduced hepatic fibrosis and portal pressure despite only a week of therapy, which was associated with improved vasodilatory responses to acetylcholine and increased bioavailability of nitric oxide (a vaso-relaxing compound) in endothelial cells from treated mice. These findings are very appealing because fibrates are already widely prescribed to patients with hyperlipidemia, and therefore their safety is established. Human trials would seem like a logical next step. However, the results really represent an initial series of observations that merit further exploration first. Specifically, the cellular targets of PPARα actions are not clarified, as the effects could represent solely the direct impact of fenofibrate on endothelial cells, or more likely the added effects of the drug on PPARα activity in hepatocytes and/or macrophages, which also express PPARα, but probably not on hepatic stellate cells, since prior studies indicate that they are not responsive to PPARα ligands [9Everett L. Galli A. Crabb D. The role of hepatic peroxisome proliferator-activated receptors (PPARs) in health and disease.Liver. 2000; 20: 191-199Crossref PubMed Scopus (109) Google Scholar, 10Miyahara T. Schrum L. Rippe R. Xiong S. Yee Jr., H.F. Motomura K. et al.Peroxisome proliferator-activated receptors and hepatic stellate cell activation.J Biol Chem. 2000; 275: 35715-35722Crossref PubMed Scopus (426) Google Scholar]. How, then, does PPARα activation reduce fibrosis? Based on the recent study by Xie et al. [[5]Xie G, Wang X, Wang L, Atkinson RD, Kanel GC, Gaarde WA, et al. Role of differentiation of liver sinusoidal endothelial cells in progression and regression of hepatic fibrosis in rats. Gastroenterology, in press. [Epub ahead of print].Google Scholar], an attractive hypothesis is that restoration of normal endothelial responses (i.e. reversal of capillarization) drives quiescence of stellate cells through an unknown mechanism. This would imply that in doing so, net protease activity is increased to help resorb accumulated scar and reduce fibrogenic gene expression. These effects of PPARα are likely to be indirect through induction of PPARα-regulated genes, either in endothelial cells, Kupffer cells and/or hepatocytes. The most elegant approach to sort out these possibilities would be to generate mice with cell-specific knockout of PPARα. Cell-specific depletion could be accomplished by using a Cre-lox strategy in which mice expressing Cre recombinase driven by a cell type-specific promoter (e.g. albumin-Cre for hepatocytes), could be crossed with animals in which the PPARα allele is flanked by LoxP sites (i.e. ‘floxed’). This would lead in the progeny to Cre-mediated recombination that deletes PPARα only in cells in which the promoter that is driving Cre expression is active. Such a strategy would enable investigators to determine in which cell type(s) fenofibrate is exerting its actions not only in regulating vasoregulation, which appears to be a direct effect on sinusoidal endothelium, but also in fibrosis, in which the effect on stellate cells is likely to be indirect. Based on the Rodríguez-Villarupla study and related papers, it is clear that the sinusoidal endothelial cell represents a fertile focus for more studies whose findings could directly improve treatments for liver disease. Although culture and characterization methods have been more challenging for this cell type, new tools are emerging to overcome these obstacles, including an immortalized murine sinusoidal endothelial cell line [[11]Huebert R.C. Jagavelu K. Liebl A.F. Huang B.Q. Splinter P.L. LaRusso N.F. et al.Immortalized liver endothelial cells: a cell culture model for studies of motility and angiogenesis.Lab Invest. 2010; 90: 1770-1781Crossref PubMed Scopus (46) Google Scholar], identification of new cell surface markers, and characterization of endothelial cell-specific promoters [[12]Ding B.S. Nolan D.J. Butler J.M. James D. Babazadeh A.O. Rosenwaks Z. et al.Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration.Nature. 2010; 468: 310-315Crossref PubMed Scopus (547) Google Scholar]. In addition to their role in vasoregulation, sinusoidal endothelium are also a driving force behind angiogenesis in liver development, regeneration, and cancer [[13]Fernandez M. Semela D. Bruix J. Colle I. Pinzani M. Bosch J. Angiogenesis in liver disease.J Hepatol. 2009; 50: 604-620Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar]. They should no longer be viewed as a silent partner in liver homeostasis. Conflict of interestThe author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.