D. Montgomery Bissell

Noninvasive measures of liver fibrosis

As novel therapies for liver fibrosis evolve, non‐invasive measurement of liver fibrosis will be required to help manage patients with chronic liver disease. Although liver biopsy is the current and time‐honored gold standard for measurement of liver fibrosis, it is poorly suited to frequent monitoring because of its expense and morbidity, and its accuracy suffers from sampling variation. At the current writing, serum markers and imaging methods are available and increasingly in use as alternatives to biopsy. However, many questions remain about their indications, accuracy, and cost‐effectiveness, and more investigation is required before they are put into widespread use. The development of safe, inexpensive, and reliable noninvasive fibrosis measurement tools remains a research priority in clinical hepatology. (Hepatology 2006;43:S113–S120.)

Drug Metabolism in Adult Rat Hepatocytes in Primary Monolayer Culture

Primary monolayer culture of adult rat hepatocytes represents a novel and potentially useful technique to study many aspects of hepatic physiology for extended periods of time in vitro (J Cell Biol 59:722-734, 1973). In examining the hepatic drug-metabolizing system in these cells, we have discovered that the conditions of cell culture exert rapid, selective, and reproducible changes in microsomal enzymes. In the 24- to 48-hr period immediately following preparation and culture of the isolated parenchymal cells, the level of the drug-binding microsomal hemoprotein, cytochrome P-450, measured in extracts of cell homogenates, declined to less than 20% of its in vivo level, whereas NADPH-cytochrome c reductase activity was only moderately reduced and glucose-6-phosphatase activity remained unchanged. The activity of aminopyrine-N-demethylase and aniline hydroxylase also fell, paralleling the level of cytochrome P-450. By contrast, p-nitroanisole O-demethylase activity was unchanged in the cultured hepatocytes despite evidence (type I binding spectrum, NADPH requirement, inhibition by carbon monoxide or by SKF 525A) that p-nitroanisole O-demethylase is a cytochrome P-450-dependent enzyme. In culture, as in vivo, aromatic polycyclic hydrocarbons stimulated p-nitroanisole O-demethylase and aryl hydrocarbon (benzo [a] pyrene) hydroxylase activities; however, this effect was unaccompanied by a detectable increase in total carbon monoxide-binding hemoprotein. The data indicate that the profile of microsomal oxidase enzymes and their control undergo striking changes as hepatocytes adapt to cell culture.

Cytochrome P-450 heme and the regulation of hepatic heme oxygenase activity☆

Abstract The degradation of cytochrome P-450 heme in the liver has been studied by a new approach. In rats, hepatic heme was labeled by administration of a tracer pulse of [5- 14 C]δ-aminolevulinic acid (ALA), and its degradation was analyzed in terms of labeled carbon monoxide ( 14 CO) excretion, which is a specific degradation product of the labeled heme. Within minutes after administration of [5- 14 C]ALA, 14 CO was detectable and increased after 2 h to an “early peak,” reflecting the elimination of labeled heme from a rapidly turning over pool in the liver. Beyond the early peak, the rate of 14 CO production decreased in a log-linear manner, consistent with the degradation of heme in stable hepatic hemoproteins. From the rate at which 14 CO production declined during this phase, from the predominant labeling of cytochrome P-450 heme by the administered [5- 14 C]ALA and from the known turnover characteristics of this hemoprotein in the liver, it could be inferred that production of 14 CO—between 16 and 30 h after administration of labeled ALA—largely reflected degradation of cytochrome P-450 heme. This approach, which permits serial measurements in a single animal, was used to study the effect on cytochrome P-450 heme of administered heme or endotoxin, both of which are potent stimulators of hepatic heme oxygenase activity. Both of these substances caused marked acceleration of the degradation of cytochrome P-450 heme, the effect occurring over the same dose range as that for stimulation of hepatic heme oxygenase. The findings suggest that stimulation of this enzyme activity in the liver is closely related to the rate of degradation of cytochrome P-450 heme.

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.

Role of αvβ6 Integrin in Acute Biliary Fibrosis

Acute biliary obstruction leads to periductal myofibroblasts and fibrosis, the origin of which is uncertain. Our study provides new information on this question in mice and humans. We show that bile duct obstruction induces a striking increase in cholangiocyte αvβ6 integrin and that expression of this integrin is directly linked to fibrogenesis through activation of transforming growth factor beta (TGF‐β). Administration of blocking antibody to αvβ6 significantly reduces the extent of acute fibrosis after bile duct ligation. Moreover, in β6‐null mice subjected to the injury, fibrosis is reduced by 50% relative to that seen in wild‐type mice, whereas inflammation occurs to the same extent. The data indicate that αvβ6, rather than inflammation, is linked to fibrogenesis. It is known that αvβ6 binds latent TGF‐β and that binding results in release of active TGFβ. Consistent with this, intracellular signaling from the TGFβ receptor is increased after bile duct ligation in wild‐type mice but not in β6−/− mice, and a competitive inhibitor of the TGFβ receptor type II blocks fibrosis to the same extent as antibody to αvβ6. In a survey of human liver disease, expression of αvβ6 is increased in acute, but not chronic, biliary injury and is localized to cholangiocyte‐like cells. Conclusion: Cholangiocytes respond to acute bile duct obstruction with markedly increased expression of αvβ6 integrin, which is closely linked to periductal fibrogenesis. The findings provide a rationale for the use of inhibitors of αvβ6 integrin or TGFβ for down‐regulating fibrosis in the setting of acute or ongoing biliary injury. (HEPATOLOGY 2007.)

Formation of extracellular matrix in normal rat liver: Lipocytes as a major source of proteoglycan

Proteoglycans are a major component of the normal hepatic extracellular matrix and undergo quantitative and qualitative changes in hepatic fibrosis. The cellular sources of proteoglycans are as yet incompletely defined. We examined this question using primary cultures of hepatocytes and lipocytes isolated from normal rat liver. Proteoglycan synthesis was assessed by measuring production of sulfated glycosaminoglycan, the polysaccharide moiety of proteoglycans. The findings indicate that lipocytes produce sixfold more glycosaminoglycan, per cell, than do hepatocytes. Two-thirds of the newly synthesized material is cell- or matrix-associated. Of the individual glycosaminoglycan species produced by lipocytes, dermatan sulfate represents 60% of the total; heparan sulfate and chondroitin sulfate are measurable but relatively minor. In hepatocyte cultures, heparan sulfate accounted for essentially all of the glycosaminoglycan detected. We conclude that lipocytes are an important source of proteoglycan in normal liver and may be the principal source of dermatan sulfate associated with hepatic fibrosis.

Hepatic fibrosis 2006: Report of the third AASLD Single Topic Conference

The third American Associated for the Study of Liver Diseases (AASLD)–sponsored Single Topic Conference on hepatic fibrosis was held in June 2006. The conference was both international, with 6 countries represented, and cross‐disciplinary, linking the basic molecular and cellular biology of fibrogenic cells to clinical trial design for emerging antifibrotic therapies. The specific goals of the conference were: (1) to consolidate knowledge about the natural history of fibrosis; (2) to clarify potential endpoints and markers; (3) to emphasize new antifibrotic targets developed on the basis of advances in basic science; and (4) to understand current critical issues pertaining to clinical trial design. Given the tremendous growth of the field and the constraints of a 2‐day format, the selection of speakers was a challenge. A number of topics not included in the oral presentations were featured at poster sessions, lending breadth and depth to the meeting as a whole. Surprising new themes emerged about molecular, clinical, and regulatory aspects of the field, and a consensus emerged that hepatic fibrosis has matured into an integrated discipline that promises to significantly improve the prognosis of patients with fibrosing liver disease. (HEPATOLOGY 2007;45:242–249.)

Transforming Growth Factor-β Initiates Wound Repair in Rat Liver through Induction of the EIIIA-Fibronectin Splice Isoform

A prominent feature of the hepatic response to injury is production of a fetal isoform of fibronectin, a splice variant containing the EIIIA region, which appears very early after injury and derives from sinusoidal endothelial cells. Previous studies have shown that it is instrumental in initiating the cellular response to injury, specifically the conversion of resting stellate cells to myofibroblast-like cells. The present work describes the regulation of this change in fibronectin expression. Using sinusoidal endothelial cells from normal or injured liver in primary culture, we show that exogenous transforming growth factor beta (TGFbeta) stimulates [EIIIA]Fn expression. To assess the role of TGFbeta in vivo, we used a chimeric IgG containing the extracellular portion of the TGFbeta type II receptor as a competitive inhibitor of the cytokine. Administered to animals at the time of injury, the inhibitor reduced expression of [EIIIA]Fn mRNA by 50% as compared to controls (P < 0.01). There was a corresponding decrease in [EIIIA]Fn protein production as judged by immunohistochemistry. Cell fractionation experiments indicated that the changes observed in whole-liver extracts were localized to sinusoidal endothelial cells. We conclude that TGFbeta initiates wound repair in part by stimulating endothelial expression of [EIIIA]Fn. Results with the soluble inhibitor of the TGFbeta type II receptor suggest a novel strategy for modulating wound repair in vivo.

Cytochrome P-450 heme and the regulation of δ-aminolevulinic acid synthetase in the liver

Abstract Hepatic δ-aminolevulinic acid synthetase was induced in rats injected with allylisopropylacetamide. The induction process was studied in relation to experimental perturbation of cytochrome P-450 in the liver. Animals were treated with either administered endotoxin or exogenous heme, both of which accelerate degradation of cytochrome P-450 heme. These manipulations were effective in blocking induction of δ-aminolevulinic acid synthetase, and the effect of each compound was proportional to its ability to stimulate degradation of cytochrome P-450 heme. The findings suggest that the heme moiety of cytochrome P-450 dissociates reversibly from its apoprotein and, prior to its degradation, mixes with endogenously synthesized heme to form a pool that regulates δ-aminolevulinic acid synthetase activity. A similar or identical heme fraction appears to mediate stimulation of heme oxygenase, which suggests that the regulation of δ-aminolevulinic acid synthetase and of heme oxygenase in the liver are closely interrelated.

Acute Porphyrias in the USA: Features of 108 Subjects from Porphyrias Consortium

BACKGROUND Recent descriptions of the clinical and laboratory features of subjects with acute porphyrias in the US are lacking. Our aim was to describe clinical, biochemical, and genetic features of 108 subjects. METHODS Between September 2010 and December 2012, 108 subjects with acute porphyrias (90 acute intermittent porphyrias, 9 hereditary coproporphyrias, 9 variegate porphyrias) were enrolled into an observational study. Genetic testing was performed at a central genetic testing laboratory and clinical information entered into a central database. Selected features were compared with data for adults in the US. RESULTS Most subjects (88/108, 81%) were female, with self-reported onset of symptoms in the second through fourth decades of life. The most common symptom was abdominal pain. Appendectomies and cholecystectomies were common before a diagnosis of porphyria. The diagnosis was delayed by a mean of 15 years. Anxiety and depression were common, and 18% complained of chronic symptoms, especially neuropathic and other pains. The incidences of systemic arterial hypertension, chronic kidney disease, seizure disorders, and psychiatric conditions were markedly increased. Mutations of the known causative genes were found in 102/105 of those tested, with novel mutations being found in 37, including in 7/8 subjects with hereditary coproporphyria. Therapy with intravenous hematin was the most effective therapy both for treatment of acute attacks and for prevention of recurrent attacks. CONCLUSIONS Acute porphyrias often remain undiagnosed for more than a decade after first symptoms develop. Intravenous hematin is the treatment of choice, both for treatment of acute attacks and for prevention of recurrent attacks.