Jacquelyn J. Maher

Support of cultured hepatocytes by a laminin-rich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver.

The subendothelial space of normal rat liver contains the constituent proteins of a basal lamina, as judged by immunohistochemical study of tissue sections. However, it is unknown whether these proteins constitute a complex with effects on hepatocellular function. We have examined this question, using normal rat hepatocytes cultured on substrata of matrix proteins as a model of the interaction between cells and basal lamina in vivo. In cultures on a type I collagen substratum, albumin secretion decreased progressively after 2 d. By contrast, when cells were cultured on a laminin-rich gel matrix, albumin secretion was stable for at least 3 wk; other functions and ultrastructural morphology were similarly maintained. None of the individual matrix proteins effectively substituted for the gel matrix, suggesting that full support of hepatocellular function requires a complex of matrix proteins. We speculate that a cause of hepatocellular dysfunction in acute inflammation is disruption of this matrix and alteration of its interaction with the hepatocyte plasma membrane.

Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo.

Whether parenchymal or nonparenchymal liver cells play a predominant role in the pathophysiology of hepatic fibrosis has not been firmly established in vivo. We have addressed this question by quantitating the relative abundance of specific mRNAs for collagen types I, III, and IV, and laminin in purified populations of hepatocytes, sinusoidal endothelial cells, and lipocytes from normal and fibrotic rat liver. In normal liver, type I collagen gene expression was minimal in all cell types; mRNA for types III and IV collagen were apparent in endothelial cells and lipocytes, but not in hepatocytes. Laminin mRNA was present in all cell types. Induction of fibrogenesis by either bile duct ligation or carbon tetrachloride administration was associated with a substantial increase in mRNA for types I and III collagen in nonparenchymal cells. Lipocytes from fibrotic animals exhibited a greater than 30-fold increase in type I collagen mRNA relative to normal lipocytes, and greater than 40-fold relative to hepatocytes. Type III collagen mRNA reached 5 times that in normal lipocytes and greater than 120 times that in hepatocytes. Endothelial cells exhibited an isolated increase in type I collagen mRNA, reaching five times that in normal liver. Type IV collagen and laminin gene expression were not significantly increased in nonparenchymal cells during fibrogenesis; in fact, mRNA for type IV collagen and laminin decreased by up to 50% in endothelial cells. Despite the pronounced changes that occurred in matrix gene expression in nonparenchymal cells during fibrogenesis, no change was noted in hepatocytes. We conclude that nonparenchymal liver cells, particularly lipocytes, are important effectors of hepatic fibrosis in vivo.

Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs.

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding αv integrin subunit because various αv-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride–induced hepatic fibrosis, whereas global loss of β3, β5 or β6 integrins or conditional loss of β8 integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the αv integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of αv-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all αv integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.

Mice fed a lipogenic methionine-choline-deficient diet develop hypermetabolism coincident with hepatic suppression of SCD-1

Lipogenic diets that are completely devoid of methionine and choline (MCD) induce hepatic steatosis. MCD feeding also provokes systemic weight loss, for unclear reasons. In this study, we found that MCD feeding causes profound hepatic suppression of the gene encoding stearoyl-coenzyme A desaturase-1 (SCD-1), an enzyme whose regulation has significant effects on metabolic rate. Within 7 days of MCD exposure, hepatic SCD-1 mRNA decreased to nearly undetectable levels. By day 21, SCD-1 protein was absent from hepatic microsomes and fatty acids showed a decrease in monounsaturated species. These changes in hepatic SCD-1 were accompanied by signs of hypermetabolism. Calorimetry revealed that MCD-fed mice consumed 37% more energy than control mice (P = 0.0003). MCD feeding also stimulated fatty acid oxidation, although fatty oxidation genes were not significantly upregulated. Interestingly, despite their increased metabolic rate, MCD-fed mice did not increase their food consumption, and as a result, they lost 26% of their body weight in 21 days. In summary, MCD feeding suppresses SCD-1 in the liver, which likely contributes to hypermetabolism and weight loss. MCD feeding also induces hepatic steatosis, by an independent mechanism. Viewed together, these two disparate consequences of MCD feeding (weight loss and hepatic steatosis) give the appearance of an unusual form of lipodystrophy.

Beyond insulin resistance: Innate immunity in nonalcoholic steatohepatitis

Obesity is an inflammatory disorder characterized by heightened activity of the innate immune system. Innate immune activation is central to the development of obesity‐related insulin resistance; it also plays an important role in obesity‐related tissue damage, such as that seen in atherosclerosis. Recent research has implicated the innate immune system in the pathophysiology of obesity‐related liver disease. This review summarizes how innate immune processes, occurring both within and outside the liver, cause not only insulin resistance but also end‐organ damage in the form of nonalcoholic fatty liver disease. (HEPATOLOGY 2008;48:670–678.)

VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis

Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60–75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1α–independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte–endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.NOTE: In the version of this article initially published, the name of one of the authors, Nihar R. Nayak, was misspelled as Nihar R. Niyak. The error has been corrected in the HTML and PDF versions of the article.

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.

Cell-specific expression of hepatocyte growth factor in liver. Upregulation in sinusoidal endothelial cells after carbon tetrachloride.

The cellular origin of hepatocyte growth factor (HGF), a polypeptide implicated in liver regeneration, was examined in normal liver and in hepatic regeneration induced by carbon tetrachloride. In normal liver, HGF and its mRNA were abundant in lipocytes, with smaller amounts present also in sinusoidal endothelial and Kupffer cells. In regenerating liver, HGF gene expression increased exclusively in endothelial cells. HGF mRNA levels rose sixfold in these cells, peaking at 6 h after toxin administration and returning to near normal by 24 h. The rise in HGF mRNA was accompanied by a 5.4-fold increase in HGF secretion. CCl4 did not alter HGF expression by either Kupffer cells or lipocytes; nor did it induce HGF expression by hepatocytes. Nonparenchymal liver cells contained two HGF transcripts: one predicting a full-length molecule of 728 amino acids; and the other encoding a functional five-amino acid deletion variant of HGF. The variant was less abundant than the full-length transcript, but increased in parallel with native HGF mRNA in response to CCl4. The response of nonparenchymal cells to HGF was examined by plating endothelial cells and lipocytes in the presence of recombinant human HGF. Under the conditions examined, the growth factor exerted neither mitogenic nor scatter factor activity on these cells.

Bile duct ligation in rats induces biliary expression of cytokine-induced neutrophil chemoattractant

BACKGROUND & AIMS Bile duct obstruction causes neutrophilic inflammation of the liver and leads to hepatic fibrosis. In obstructive liver disease, the localization of neutrophils in portal tracts suggests that cells within this region produce neutrophil chemoattractants. In this study, we investigated whether bile duct obstruction in rats induces portal expression of cytokine-induced neutrophil chemoattractant (CINC). METHODS Rats underwent bile duct ligation for 3 hours to 8 days. CINC regulation was examined in vivo at various intervals by immunohistochemistry, ribonuclease protection, and in situ hybridization. CINC production was also investigated in cell culture, in response to putative stimuli from obstructed liver. RESULTS Bile duct ligation caused neutrophilic infiltration of the liver within 3 hours. CINC was also rapidly induced, with specific expression identified in biliary cells. Rat intrahepatic biliary cells produced CINC constitutively in culture; when exposed to cholestatic bile, they showed a 12-fold increase in CINC secretion. The effect of bile was not attributable to toxicity or to dissolved cytokines or endotoxin. Mechanical strain, designed to mimic the stretching of biliary cells during obstruction, did not induce CINC. CONCLUSIONS Biliary cells contribute to hepatic inflammation during cholestasis by producing neutrophil chemoattractants. A major stimulus to biliary chemoattractant production in vivo may be bile itself.

Nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a disorder characterized by excess accumulation of fat in hepatocytes (nonalcoholic fatty liver (NAFL)); in up to 40% of individuals, there are additional findings of portal and lobular inflammation and hepatocyte injury (which characterize nonalcoholic steatohepatitis (NASH)). A subset of patients will develop progressive fibrosis, which can progress to cirrhosis. Hepatocellular carcinoma and cardiovascular complications are life-threatening co-morbidities of both NAFL and NASH. NAFLD is closely associated with insulin resistance; obesity and metabolic syndrome are common underlying factors. As a consequence, the prevalence of NAFLD is estimated to be 10–40% in adults worldwide, and it is the most common liver disease in children and adolescents in developed countries. Mechanistic insights into fat accumulation, subsequent hepatocyte injury, the role of the immune system and fibrosis as well as the role of the gut microbiota are unfolding. Furthermore, genetic and epigenetic factors might explain the considerable interindividual variation in disease phenotype, severity and progression. To date, no effective medical interventions exist that completely reverse the disease other than lifestyle changes, dietary alterations and, possibly, bariatric surgery. However, several strategies that target pathophysiological processes such as an oversupply of fatty acids to the liver, cell injury and inflammation are currently under investigation. Diagnosis of NAFLD can be established by imaging, but detection of the lesions of NASH still depend on the gold-standard but invasive liver biopsy. Several non-invasive strategies are being evaluated to replace or complement biopsies, especially for follow-up monitoring.