Methods for the study of liver cell heterogeneity

A large number of histological, histochemical and biochemical techniques are available for studying liver cell heterogeneity. Structural differences are recognized by morphometric analyses of electron micrographs. The zonal heterogeneity of enzyme activities can be demonstrated by histochemistry and more precisely by ultramicrobiochemical assays in microdissected periportal and perivenous tissue. Immunohistochemistry is useful for quantifying and localizing proteins, especially isoenzymes, without depending on their biological activity. The zonal quantification of specific mRNA can be achieved by in situ hybridization. The different structural and enzymic equipment of periportal and perivenous tissue found by these techniques has led to the concept of metabolic zonation. This hypothesis can be confirmed by determination of metabolic rates in perfused liver after selective zonal damage, in separated periportal and perivenous hepatocytes as well as in periportal and perivenous tissue of perfused liver by non-invasive techniques.     

Quantitative and qualitative histochemical investigation on NADP+-dependent dehydrogenases in the limiting plate and the residual parenchyma surrounding terminal hepatic venules

Activities of three NADP+-dependent enzymes (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase) were demonstrated in the first layer of hepatocytes adjacent to terminal hepatic venules (perivenous limiting plate), and in the residual parenchyma of the perivenous zone of the acinus, in normally fed adult male Wistar rats, using a Lowry technique and a qualitative histochemical staining reaction. Enzyme activities of the glucose-6-phosphate dehydrogenase were significantly higher in the hepatocytes adjacent to terminal hepatic venules (ratio hepatocytes adjacent to terminal hepatic venules/residual parenchyma of the perivenous zone: 1.31). 6-Phosphogluconate dehydrogenase and isocitrate dehydrogenase were homogeneously distributed in the two areas measured (ratio: 1.04 and ratio: 1.0 respectively). With the qualitative histochemical staining reactions no differences were found.     

Quantitative and qualitative histochemical investigation on NADP+-dependent dehydrogenases in the limiting plate and the residual parenchyma surrounding terminal hepatic venules

Summary Activities of three NADP⁺-dependent enzymes (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase) were demonstrated in the first layer of hepatocytes adjacent to terminal hepatic venules (perivenous limiting plate), and in the residnal parenchyma of the perivenous zone of the acinus, in normally fed adult male Wistar rats, using a Lowry technique and a qualitative histochemical staining reaction. Enzyme activities of the glucose-6-phosphate dehydrogenase were significantly higher in the hepatocytes adjacent to terminal hepatic venules (ratio hepatocytes adjacent to terminal hepatic venules/residual parenchyma of the perivenous zone: 1.31). 6-Phosphogluconate dehydrogenase and isocitrate dehydrogenase were homogeneously distributed in the two areas measured (ratio: 1.04 and ratio: 1.0 respectively). With the qualitative histochemical staining reactions no differences were found.Supported by the Deutsche Forschungsgemeinschaft (Hi318/2-1)     

Superoxide mediates endotoxin-induced platelet-endothelial cell adhesion in intestinal venules

Platelets have been implicated in the pathogenesis of different diseases of the vascular system, including atherosclerosis, sepsis, and ischemia-reperfusion injury; however, relatively little is known about the factors that regulate the interactions between circulating platelets and the vessel wall. The objective of this study was to define the contribution of superoxide to LPS-induced platelet-endothelial cell (P/E) adhesion in murine intestinal venules. The adhesion of rhodamine-6G-labeled murine platelets was monitored by intravital fluorescence microscopy. Four hours after LPS administration in control [wild-type (WT)] mice, an approximately 10-fold increase in P/E adhesion was detected. This response did not result from LPS-induced platelet activation. The LPS-induced P/E adhesion was greatly attenuated in NAD(P)H oxidase-deficient mice and in WT mice rendered neutropenic with anti-neutrophil serum, whereas the response was unchanged in WT mice receiving a CD18 blocking MAb or in CD18-deficient mice. A chimeric form of MnSOD that exhibits the binding properties of extracellular SOD also attenuated the LPS-induced response in WT mice. These findings indicate that neutrophil-derived superoxide plays a major role in the modulation of endotoxin-induced P/E adhesion.     

Pancreatic islet cell tumors metastatic to the liver: treatment by hepatic artery chemo-embolization

The embolization of hepatic metastases of pancreatic islet cell tumors achieves a tumor necrosis without liver failure owing to double hepatic blood supply. The arterial chemotherapy performed at the same time delivers a large amount of cytotoxic agent directly into the tumor. Tumor bulk reducing and decreasing of production of pharmacologically active substances may be obtained to some degree. Although an objective documentation of the therapeutic effect is difficult to obtain, several series emphasize that the embolization provides a partial and transient palliative remission. Therefore, the chemo-embolization should be included in a multidisciplinary approach of metastatic endocrine malignancies in combination with surgical resection, systemic antineoplastic chemotherapy, and antihormonal therapy.     

An iterative approach to placing counterions around DNA

An iterative approach, in which the effect of placing counter ions around DNA influences the electrostatic potential that the other subsequently approaching ions feel, has been used to place sodium ions around polynucleotides. The main focus of this report is to study the sequence and structure dependence on the distribution of ions around DNA, particularly that of tightly bound ions. The interesting results of the calculations are that there is significant sequence dependence on the electrostatic potentials in the B form of DNA, whereas relatively less difference in A form. In the case of Z form, the cations bridge the inter-stand phosphates along the minor groove.     

A non-parametric approach to translating gene region heterogeneity associated with phenotype into location heterogeneity

MOTIVATION: The analysis of genetic data poses statistical problems in the form of high dimensionality with small sample sizes. The construction of a composite gene region (sequence pair) heterogeneity measure is one technique for reducing the dimensionality of the problem. This approach however is not without cost, since the contribution of locations to observed gene region differences between groups becomes entangled in this summary measure. This is problematic since it is of scientific interest to identify locations that together depict phenotype. RESULTS: A method is proposed for relating observed gene region heterogeneity back to the location level. In the spirit of a factor analysis-type setting, the approach focuses on identifying a latent variable structure among locations to explain within and between group genetic differences associated with phenotype. The method is flexible for identifying either the additive contribution from individual locations or the additive contribution from a group of locations, to observed gene region heterogeneity, depending upon the weighting scheme used in constructing a gene region heterogeneity measure. The approach is illustrated with clinical trial data, where the problem of altered HIV drug susceptibility is examined through characterizing location contributions to HIV protease gene region differences associated with a phenotypic treatment response. AVAILABILITY: The Splus (MathSoft, Inc. S-Plus 2000, Seattle, WA, 1999) developed menu-driven functions for obtaining results, GENE_ S (J.Kowalski, Harvard School of Public Health, Boston, MA 2001), is available from the author upon request.     

Allogeneic hepatic cell suspension in the treatment of liver insufficiency

A study was made of a possibility of using isolated hepatocytes for the treatment of diseases of the liver in animals. The optimal dose of the cell suspension in intravascular, intraperitoneal, intrapleural and subcutaneous administration was determined; a reaction of the experimental animals to this biological substrate was studied. The efficacy of the isolated hepatocytes in the treatment of hepatic insufficiency is demonstrated; also a comparative assessment of the mentioned methods of the cell suspension administration is given.     

Apparent heterogeneity of hepatic lysosomes due to membrane-bound acid phosphatase.

Hepatic lysosomes have been fractionated by rate sedimentation and by isopycnic banding. In all experiments, the distribution of acid phosphatase differed from that of the other lysosomal enzymes. Evidence is presented that this difference is due not to the separation of lysosomes from different cell types, but simply reflects the membrane location of a part of the acid phosphatase.     

A new approach to an unbiased estimate of the hepatic stellate cell index in the rat liver: an example in healthy conditions.

Evaluation of activation and proliferation of hepatic stellate cells (HSCs) must be grounded on solid quantitative data under normal conditions. The HSC index (HSCI), number of HSCs per 1000 hepatocytes (HEP), is often used in hepatology but has been never determined using stereology. Systematically sampled sections were immunostained against glial fibrillary acidic protein and carcinoembryonic antigen, allowing unequivocal distinction of HSC and mononuclear/binuclear HEP. With the optical disector the HSCI was estimated as 109 (coefficient of error = 0.04). This work provides a sound technical basis for experiments in which the estimation of HSCI and/or simultaneous quantification of HSC and HEP are relevant.     

Mast cell heterogeneity

Increasing evidence for the existence of inter- and intra-species mast cell heterogeneity has expanded the potential biological role of this cell. Early studies suggesting that mast cells at mucosal sites differ morphologically and histochemically from connective tissue mast cells have been confirmed using isolated intestinal mucosal mast cells in the rat and more recently in man. These studies also established that mucosal mast cells are functionally distinct from connective tissue mast cells. Thus, mucosal and connective tissue mast cells differ in their responsiveness to a variety of mast cell secretagogues and antiallergic agents. Speculation about the therapeutic use of antiallergic drugs in disorders involving intestinal mast cells cannot, therefore, be based on extrapolation from studies of their effects on mast cells from other sites. Regulatory mechanisms for mast cell secretion may also be heterogeneous since mucosal mast cells differ from connective tissue mast cells in their response to a variety of physiologically occurring regulatory peptides. The development of techniques to purify isolated mast cell subpopulations will facilitate future analysis of the biochemical basis of the functional heterogeneity of mast cells.     

Primary perivascular epithelioid cell tumor of the liver not related to hepatic ligaments: hepatic PEComa as an emerging entity

Primary perivascular epithelioid cell tumor (PEComa) of the liver is a very rare example of an emerging family of hepatic PEC tumors. Only few cases have been described so far. We report the case of a large but benign hepatic PEComa in a 53-year-old man without signs of tuberous sclerosis. In contrast to recently described PEC-derived liver tumors in children and young adults, this neoplasm was not related to the hepatic ligaments but had developed deeply within the liver substance. The neoplastic cells displayed the complete phenotype typical for PEComas, i.e. reactivity for several melanoma markers and for smooth muscle actin. The unique relationship of myoid tumor cells to the adventitia of blood vessels prompted us, in comparison with published findings obtained with angiomyolipomas, to comment on the possible origin of the still enigmatic perivascular epithelioid cells.     

Single cell heterogeneity

Multi-level heterogeneity is a fundamental but underappreciated feature of cancer. Most technical and analytical methods either completely ignore heterogeneity or do not fully account for it, as heterogeneity has been considered noise that needs to be eliminated. We have used single-cell and population-based assays to describe an instability-mediated mechanism where genome heterogeneity drastically affects cell growth and cannot be accurately measured using conventional averages. First, we show that most unstable cancer cell populations exhibit high levels of karyotype heterogeneity, where it is difficult, if not impossible, to karyotypically clone cells. Second, by comparing stable and unstable cell populations, we show that instability-mediated karyotype heterogeneity leads to growth heterogeneity, where outliers dominantly contribute to population growth and exhibit shorter cell cycles. Predictability of population growth is more difficult for heterogeneous cell populations than for homogenous cell populations. Since “outliers” play an important role in cancer evolution, where genome instability is the key feature, averaging methods used to characterize cell populations are misleading. Variances quantify heterogeneity; means (averages) smooth heterogeneity, invariably hiding it. Cell populations of pathological conditions with high genome instability, like cancer, behave differently than karyotypically homogeneous cell populations. Single-cell analysis is thus needed when cells are not genomically identical. Despite increased attention given to single-cell variation mediated heterogeneity of cancer cells, continued use of average-based methods is not only inaccurate but deceptive, as the “average” cancer cell clearly does not exist. Genome-level heterogeneity also may explain population heterogeneity, drug resistance, and cancer evolution.     

Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease

During chronic injury a population of bipotent hepatic progenitor cells (HPCs) become activated to regenerate both cholangiocytes and hepatocytes. Here we show in human diseased liver and mouse models of the ductular reaction that Notch and Wnt signaling direct specification of HPCs via their interactions with activated myofibroblasts or macrophages. In particular, we found that during biliary regeneration, expression of Jagged 1 (a Notch ligand) by myofibroblasts promoted Notch signaling in HPCs and thus their biliary specification to cholangiocytes. Alternatively, during hepatocyte regeneration, macrophage engulfment of hepatocyte debris induced Wnt3a expression. This resulted in canonical Wnt signaling in nearby HPCs, thus maintaining expression of Numb (a cell fate determinant) within these cells and the promotion of their specification to hepatocytes. By these two pathways adult parenchymal regeneration during chronic liver injury is promoted.