Marcos Rojkind

Collagen Types in Normal and Cirrhotic Liver

The purpose of this study was to characterize the collagen in normal liver and in liver cirrhosis of different etiology, and to determine whether the ratios of different collagen types vary with the agent that induced the disease. Collagens were extracted from five normal and eight cirrhotic livers, which were obtained at autopsy. Alcoholic, hemochromatotic, Wilsons disease, and posthepatitic cirrhosis were studied. The yield of extracted collagens was 60–95%. Normal liver contained 5.5 ± 1.6 mg of collagen/ g of wet tissue. In cirrhotic liver, collagen was increased four- to sevenfold. In all livers studied, approximately 2 mg of collagen/g of wet tissue remained insoluble. Each liver contained Type I and Type III collagen, and a group of collagens with amino acid composition similar to that found in basement membranes. Three of these basement membrane collagens, A, B, and E, representing onehalf of the liver basement membrane collagen, were purified and partially characterized. The other half, similar in composition to basement membrane collagen obtained from glomerulus, was heterogeneous and was not characterized. Collagens A and Bare identical to the A and B chains isolated from placenta and skin. Collagen E is similar to the collagen previously isolated from human aorta. In cirrhotic, but not normal liver, an additional collagen, which was identified as a trimer of α1 Type I collagen, was found. Normal livers contained approximately equal amounts of Type I, Type Ill, and basement membrane collagens; The ratio of Type l/Type III collagen was smaller than 1, suggesting a slight predominance of Type III collagen. In cirrhotic livers regardless of etiology, all collagen Types were increased; in livers containing 20 mg of collagen/g of tissue, Type I collagen was the predominant type, and the ratio of Type I/Type III collagen was increased.

Colchicine in the Treatment of Cirrhosis of the Liver

There is preliminary evidence that colchicine, an inhibitor of collagen synthesis, may be beneficial in the treatment of cirrhosis of the liver. To evaluate the use of colchicine (1 mg per day, five days per week) in the treatment of hepatic cirrhosis, we performed a randomized, double-blind, placebo-controlled trial in which 100 patients were followed for up to 14 years. Forty-five patients had alcoholic cirrhosis, 41 had posthepatitic cirrhosis, and the remaining 14 had cirrhosis with various other causes. Histologic studies were available for 92 percent of patients. Seventy-three patients were in Child-Turcotte class A, 26 were in class B, and one was in class C. Fifty-four patients received colchicine, and 46 received placebo. The overall survival in the colchicine group was markedly better than in the placebo group (median survival, 11 and 3.5 years, respectively; P less than 0.001). The cumulative 5-year survival rates were 75 percent in the colchicine group and 34 percent in the placebo group; the corresponding 10-year survival rates were 56 percent and 20 percent. Among the 30 patients treated with colchicine who underwent repeated liver biopsies, histologic improvement was seen in 9; the liver appeared normal in 2, and 7 had minimal portal fibrosis. No histologic improvement was observed in the 14 members of the placebo group who had two or more biopsies. Few side effects were observed in either group.

Hedgehog pathway activation and epithelial-to-mesenchymal transitions during myofibroblastic transformation of rat hepatic cells in culture and cirrhosis

Myofibroblastic hepatic stellate cells (MF-HSC) are derived from quiescent hepatic stellate cells (Q-HSC). Q-HSC express certain epithelial cell markers and have been reported to form junctional complexes similar to epithelial cells. We have shown that Hedgehog (Hh) signaling plays a key role in HSC growth. Because Hh ligands regulate epithelial-to-mesenchymal transition (EMT), we determined whether Q-HSC express EMT markers and then assessed whether these markers change as Q-HSC transition into MF-HSC and whether the process is modulated by Hh signaling. Q-HSC were isolated from healthy livers and cultured to promote myofibroblastic transition. Changes in mRNA and protein expression of epithelial and mesenchymal markers, Hh ligands, and target genes were monitored in HSC treated with and without cyclopamine (an Hh inhibitor). Studies were repeated in primary human HSC and clonally derived HSC from a cirrhotic rat. Q-HSC activation in vitro (culture) and in vivo (CCl(4)-induced cirrhosis) resulted in decreased expression of Hh-interacting protein (Hhip, an Hh antagonist), the EMT inhibitors bone morphogenic protein (BMP-7) and inhibitor of differentiation (Id2), the adherens junction component E-cadherin, and epithelial keratins 7 and 19 and increased expression of Gli2 (an Hh target gene) and mesenchymal markers, including the mesenchyme-associated transcription factors Lhx2 and Msx2, the myofibroblast marker alpha-smooth muscle actin, and matrix molecules such as collagen. Cyclopamine reverted myofibroblastic transition, reducing mesenchymal gene expression while increasing epithelial markers in rodent and human HSC. We conclude that Hh signaling plays a key role in transition of Q-HSC into MF-HSC. Our findings suggest that Q-HSC are capable of transitioning between epithelial and mesenchymal fates.

Role for Hedgehog signaling in hepatic stellate cell activation and viability

Hepatic stellate cells (HSC) have a complex phenotype that includes both neural and myofibroblastic features. The Hedgehog (Hh) pathway has been shown to direct the fate of neural and myofibroblastic cells during embryogenesis and during tissue remodeling in adults. Therefore, we hypothesized that Hh signaling may regulate the fate of HSC in adults. In this study, we find that freshly isolated stellate cells from adult Patched-lacZ transgenic mice exhibit β-galactosidase activity, indicating Hh pathway activity. Transcripts of Hh ligands, the Hh pathway receptor, and Hh-regulated transcription factors are expressed by stellate cells from mice, rats, and humans. Transfection experiments in a cell line using a Hh-inducible luciferase reporter demonstrate constitutive Hh pathway activity. Moreover, neutralizing antibodies to Hh increase apoptosis, while viability is restored by treatment with Hh ligand. In vitro treatment of primary stellate cells with cyclopamine (Cyc), a pharmacologic inhibitor of the Hh pathway, inhibits activation and slightly decreases cell survival, while a single injection of Cyc into healthy adult mice reduces activation of HSC by more than 50% without producing obvious liver damage. Our findings reveal a novel mechanism, namely the Hh pathway, that regulates the activation and viability of HSC.

A synthetic peptide from transforming growth factor β type III receptor inhibits liver fibrogenesis in rats with carbon tetrachloride liver injury

Transforming growth factor beta1 (TGF-beta1) is a pleiotropic cytokine, which displays potent profibrogenic effects and is highly expressed in fibrotic livers. For this reason, development of TGF-B1 inhibitors might be of great importance to control liver fibrogenesis as well as other undesired side effects due to this cytokine. Potential peptide inhibitors of TGF-beta1 (derived from TGF-beta1 and from its type III receptor) were tested in vitro and in vivo using different assays. Peptides P11 and P12, derived from TGF-beta1, and P54 and P144, derived from its type III receptor, prevented TGF-beta1-dependent inhibition of MV1Lu proliferation in vitro and markedly reduced binding of TGF-beta1 to its receptors. P144 blocked TGF-beta1-dependent stimulation of a reporter gene under the control of human alpha2(I) collagen promoter. Intraperitoneal administration of P144 also showed potent antifibrogenic activity in vivo in the liver of rats receiving CCl4. These rats also showed a significant decrease in the number of activated hepatic stellate cells as compared with those treated with saline only. These results suggest that short synthetic peptides derived from TGF-beta1 type III receptor may be of value in reducing liver fibrosis in chronic liver injury.

The relationship between the free pool of proline and collagen content in human liver cirrhosis.

The free proline, free glutamic acid, and total collagen contents of the livers of cirrhotic and noncirrhotic patients were determined. The amounts of free proline in the sera of the patients were also determined. The results indicated that certain metabolic changes occurred in cirrhotic livers of humans that were similar to the metabolic changes observed previously in CCl(4)-induced cirrhosis in the rat. The amount of free proline was coordinate with the increase in total collagen, and both were inversely related to the amount of free glutamic acid. The average proline concentration in sera of cirrhotic patients was not higher than that of non cirrhotic patients, suggesting that the metabolic alteration noted above is a local event in the liver related to fibrogenesis. These and other results suggest that the pool size of free proline may play a prime role in regulation of collagen biosynthesis in liver cirrhosis.

Effect of colchicine on collagen, albumin and transferrin synthesis by cirrhotic rat liver slices

Collagen synthesis was found to be increased in liver slices of rats made cirrhotic by chronic administration of CCl4. The liver function was impaired, as determined by an increased retention of conjugated bilirubin and low serum albumin values. However, when animals received colchicine simultaneously with CCl4, collagen synthesis and deposition were inhibited, and the liver function appeared normal. When a group of rats was made cirrhotic by chronic administration of CCl4, and then kept for 30 days without further treatment, fibrosis persisted and collagen synthesis was very low. However, the liver function was severely impaired. When similar rats received L-azetidine-2-carboxylic acid during the 30-days period following CCl4 administration, there was a slight but not significant improvement in liver function. The collagen synthesis and the extent of fibrosis were similar to the controls. However, if similar rats received colchicine during the 30 days period, collagen synthesis was almost negligible, there was a slight decrease in fibrosis and there was a great improvement in liver function. In all the cirrhotic animals studied, transferrin biosynthesis remained constant.

Early response of α2(I) collagen to acetaldehyde in human hepatic stellate cells is TGF-β independent†

Acetaldehyde is fibrogenic and induces the expression of type I collagen genes in hepatic stellate cells. Some of these acetaldehyde‐dependent events are mediated by H2O2 and thus establish a direct connection between oxidative stress and collagen upregulation. We localized to the −378 to −183 region of the α2(I) collagen (COL1A2) promoter an acetaldehyde‐responsive element (AcRE) functional in human hepatic stellate cells (HHSCs) and investigated molecular mechanisms whereby acetaldehyde stimulates and modulates its transcriptional activity. Because the AcRE co‐localized with a previously described transforming growth factor β (TGF‐β)1–responsive element, and both acetaldehyde and this cytokine induce their effects through H2O2, we investigated whether all fibrogenic actions of acetaldehyde were mediated by this cytokine. Here we show that acetaldehyde‐induced COL1A2 upregulation in HHSCs recognizes two distinct but overlapping early and late stages that last from 1 to 6 hours and from 6 to 24 hours, respectively. We present several lines of evidence to show that early acetaldehyde‐mediated events are independent of TGF‐β1. These include significant time‐course differences in the expression of COL1A2 and TGF‐β1 mRNAs and inability of neutralizing antibodies to TGF‐β1 to inhibit acetaldehyde‐dependent collagen gene transcription and Smad 3 phosphorylation. We also show that although acetaldehyde‐dependent upregulation of collagen was PI3K dependent, that of TGF‐β1 was PI3K independent. In conclusion, acetaldehyde‐dependent mechanisms involved in COL1A2 upregulation are similar, but not identical, to those of TGF‐β1. We suggest that early acetaldehyde‐dependent events induce the late expression of TGF‐β1 and create an H2O2‐dependent autocrine loop that may sustain and amplify the fibrogenic response of this alcohol metabolite. (HEPATOLOGY 2005;42:343–352.)

LONG‐TERM CULTURES OF NORMAL RAT HEPATOCYTES ON LIVER BIOMATRIX

A major stumbling block in liver cell studies has been an inability to maintain functional hepatocytes in vitro for more than a few weeks.’-4 As is typical with most differentiated epithelial or epithelioid cells, hepatocytes either die or dedifferentiate in culture. We have recently proposed that such difficulties are due to the cell culture technique of dissociating a cell type away from the tissue matrix in which exists “socio-cellular” variables critical for the maintenance of the differentiated cell state.‘ These “sociocellular” variables include the components of the extracellular matrix, the basement membrane and ground substance, and the signals from cell-cell interactions (soluble signals). Therefore, to culture differentiated cells and have them retain their differentiated state, we proposed that one must simulate the crucial “sociocellular” variables in vitro. In our initial efforts to ascertain these variables, we have focused upon a ubiquitous and presumably primary cell-cell relationship, the epithelial-mesenchymal relationship. In vivo normal epithelial cells capable of proliferation or long-term survival are attached to a basement membrane which, in turn, is associated with mesenchymally-derived cells, most commonly fibroblasts. The basement membrane, a layer of secretion located between and produced by the epithelium and the mesenchymal cells, is assumed to contain the extracellular, solidstate subset of factors in the interaction. In the early studies, we used mixtures of extracellular matrix components made with purified components. Although a simplistic approach to the problem, these methods indicated that basement membrane components could significantly lengthen the life of certain differentiated cells, insulinoma cells and prostatic epithelial cells.”’ More recently, new procedures have been introduced that supercede the earlier ones and which provide methods for the isolation and use of extracellular matrix components as substrates for cell cultures.6’ The fibrous material isolated by these new procedures is referred to as biomatrix and is a complex mixture of collagens, noncollagenous proteins and carbohydrates. It can be isolated from any tissue although the yields vary significantly from one tissue to another. Tissues such as liver, with small amounts of stroma, give 1-2 gms/100 gms tissue, whereas tissues with extensive stroma or smooth muscle, such as colon or prostate, provide high yields ( 1 5-22 gms/ 100 gms tissue).

[21] New Techniques for Culturing Differential Cells: Reconstituted basement membrane rafts

Publisher Summary This chapter describes new techniques that provide routine methods for culturing differentiated cells, whether normal or malignant. The techniques are sufficiently developed and they can greatly lengthen the time that differentiated cells retain their tissue-specific functions in vitro. The need for model systems of differentiated mammalian cell types to be used in biological and clinical research is sufficiently satisfied because of the inherent difficulties in establishing differentiated cells, especially normal cells, in vitro. Analysis of these techniques and the response of cells to them reveal methodologic limitations and certain requirements for cellular differentiation and growth. Methods, which retain tissue architecture, permit retention of tissue-specific functions including hormone and pharmacological responses. However, the tissue normally degenerates within a few weeks because of difficulties in vascularizing all the cells within the tissue. There is genetic uniformity, easier maintenance of the cells, and reduction of variables associated with a multicell culture system.