Francis R. Weiner

Collagen chain mRNAs in isolated heart cells from young and adult rats

Collagen is the predominant component of the extracellular matrix of the heart, where it is organized in a hierarchy of structures. To establish the cellular origin of the various collagen types, type I-procollagen alpha 2 chain and types III and IV collagen mRNAs were examined in preparations of myocytes and non-myocyte heart cells freshly isolated from rats 1 to 6 months old. The cardiomyocytes appeared morphologically intact and functionally competent. Fibroblast-like cells predominated in the non-myocyte cell fractions but endothelial and smooth muscle cells were also present. RNA from whole ventricular tissue served as a control. Northern and dot blot analyses were used to establish the presence or absence of mRNAs. In RNA prepared from whole ventricular tissue, the mRNAs for alpha-, beta-, and gamma-actin isotypes were detected whereas mRNA for alpha-actin was found in myocytes and those for beta- and gamma-actins were found in non-myocyte cells, confirming further the nature of the cell populations. Procollagen types I and III mRNAs were not detected in the total RNA of cardiomyocytes but mRNA for type IV collagen was present. The mRNAs for all three collagen types were present in the non-myocyte cells. These results suggest that in the rat heart the non-myocyte cells, probably fibroblasts, are responsible for interstitial collagen production. Both cell populations may engage in the formation of basement membrane collagen type IV.

Increased transforming growth factor-β1 gene expression in human liver disease

We recently demonstrated that transforming growth factor-beta 1 stimulates collagen synthesis in hepatic cells in vitro, and that the synthesis of this cytokine is markedly increased in two rodent models of hepatic fibrosis. In the present study, we investigated the association of transforming growth factor-beta 1 (TFG-beta 1) gene expression in human liver disease. Sixteen patients with active liver disease had percutaneous liver biopsies performed for diagnostic purposes. Total RNA was extracted from an unused portion of each biopsy and then subjected to hybridization analysis with the following human cDNA clones: albumin, pro alpha 1 (I) collagen, and TGF-beta 1. Surgical liver biopsy specimens from two patients without hepatic disease were used as controls. When compared to controls, the patients with active liver disease had a 19% decrease in albumin, a 97% increase in type I collagen, and a 120% increase in transforming growth factor-beta 1 mRNA levels. Moreover, steady-state levels of TGF-beta 1 and procollagen mRNAs were significantly correlated. Nuclear run-on assays showed that livers from two patients with fibrosis had TGF-beta 1 transcription rates that were more than 2-fold higher than rates in control livers. These findings indicate that transforming growth factor-beta 1 gene expression is significantly enhanced in man during active liver disease.

The Effects of Hepatic Fibrosis on Ito Cell Gene Expression

While Ito cells appear to be a major source of increased matrix synthesis during hepatic fibrogenesis, the cellular changes that occur in these cells during liver fibrosis have not been well delineated. In this study we examined Ito cell gene expression in isolated cells from normal rats, and rats with carbon tetrachloride-induced fibrosis, in order to better define the changes occurring in these cells during this pathologic process. Specifically, we addressed three questions: (1) which matrix genes are over expressed in Ito cells in fibrotic liver; (2) do these cells increase their expression of the fibrogenic cytokine transforming growth factor-beta 1 (TGF-beta 1); and (3) do Ito cells change their phenotype during hepatic fibrogenesis as reflected by alterations in the expression of their intermediate filament genes? Northern hybridization analysis revealed that Ito cells isolated from fibrotic livers had significant increases in mRNA levels of types I, III and IV procollagen compared to normal cells, while no increases were found in hepatocytes, and Kupffer/endothelial cells had only an increase in type I procollagen mRNA. Analysis of other matrix proteins which increase during hepatic fibrogenesis revealed elevations in laminin B and fibronectin mRNA levels only in Ito cells. Increased Ito cell matrix gene expression was also associated with a 4-fold increase in TGF-beta 1 levels in these cells. No increase in TGF-beta 1 mRNA was found in hepatocytes, and less than a 2-fold increase was found in Kupffer/endothelial cells isolated from fibrotic livers.(ABSTRACT TRUNCATED AT 250 WORDS)

Haplotype analysis of a type I collagen gene and its association with alcoholic cirrhosis in man

Cirrhosis is a major cause of death worldwide, and in the Western hemisphere alcohol is responsible for at least 60% of deaths due to cirrhosis (Dajer et al. 1978; Galambos, 1979). However, only 8-20% of chronic ethanol abusers actually develop cirrhosis (Klatskin, 1961; Galambos, 1974). Why are some alcohol abusers predisposed to develop cirrhosis while others remain re lat ive ly unaffected? While many au thor i t i es believe that there is a genetic s u s c e p t i b i l i t y for developing cirrhosis, these studies have yielded conf l ict ing results (Conn, 1982; Maddrey, 1964; Saunders et a l . , 1982; Faizallah e t L l . , 1982; Scott e t L l . , 1977,; Bell Lt a l . , 1982; Doffoel et a l . , 1986; Monteiro e t L l . , 1986), and as yet there is no direct proof. No previous studies have used recombinant DNA techniques to address the issue of a genetic proc l iv i ty to develop alcoholic cirrhosis.

A role for cytokines as regulators of hepatic fibrogenesis

SummaryIt is evident that hepatic fibrogenesis is a complex process involving a cascade of cytokines which interact to enhance the expression of ECM. Cytokines involved early in this cascade may serve as proinflammatory agents or as stimulators of macrophage and Ito cell activation and proliferation, while those cytokines involved later in this process may be directly fibrogenic. Furthermore, we speculate that a balance between profibrogenic and antifibrogenic cytokines normally exists but in the presence of hepatic insults, a relative superabundance of the fibrogenic factors promotes the development of liver fibrosis. To date, most of the evidence supporting a role for cytokines in liver fibrosis has been obtained inin vitro systems or in animal models. We now need to extend these findings to man in order to determine whether a similar cascade of cytokines is important in the development of this pathologic process in man. Further delineation of these cytokines (as well as other profibrogenic soluble factors), and the mechanisms by which they act, are critical to our development of more rational forms of therapy for liver fibrosis.

Steroid-induced mesenteric lipomatosis

A patient undergoing chronic corticosteroid therapy presented with mesenteric lipomatosis that simulated malignant masses. Computerized tomography was instrumental in identifying these apparent tumors as benign fatty deposits. The spectrum of lipomatous abdominal lesions amenable to diagnosis by computerized tomography is reviewed.

The Effect of Ethanol on Hepatic mRNA Regulation in Baboon and Man

Serum albumin levels are frequently depressed in patients with Laennec’s cirrhosis, and it is thought that alcohol has a toxic effect on hepatic protein synthesis. However, the experimental findings are conflicting (see our review).’ With the exception of our prior work on ethanol-induced fatty liver in rats,’ no studies have addressed the effects of chronic ethanol administration on the molecular mechanisms of hepatic protein synthesis. Therefore, the primary objective of our study was to employ molecular hybridization techniques to evaluate protein synthesis in chronic alcoholic liver disease in a baboon model and then to extend our findings to man. Our second objective was to evaluate the molecular mechanisms responsible for increased collagen content in the fibrotic liver. It has been shown that in the immunological hepaptic injury of murine schistosomiasis there is a striking increase in type I ~ o l l a g e n , ~ accompanied by an increase in type I procollagen mRNA ~ o n t e n t . ~ In the present study we extended our previous work on the hepatotoxic effects of ethanol exposure by investigating the changes in type I procollagen mRNA in the livers of baboons exposed to chronic alcohol administration and in man. Using minor modifications of the procedure of Chirgwin et aL5 we extracted total RNA from percutaneous liver biopsies of five baboons who were chronically fed an ethanol-rich liquid diet, and their pair-fed controls. The R N A was then used in in-vitro protein synthesis assays4 or in RNA-DNA hybridization studies! Chronic alcohol administration in baboons with liver fibrosis and a normal serum albumin increased in-vitro protein synthesis as measured by [”SI-methionine incorporation (15.3 t 1.8 x lo4 cpm/pg RNA, in ethanol-fed vs 8.43 f 1.4 in controls; P < 0.01). Alcohol-fed baboons also had increased albumin mRNA content (180% +. 21 of control; P < 0.05) and type I procollagen mRNA content (183% ? 28 of control; P < 0.02). There was no difference in the P-actin mRNA content (a constitutive protein). We were then able to apply to man the techniques of R N A extraction developed in the baboon model. R N A was extracted from unused portions of percutaneous liver biopsies from humans having this procedure done for clinically indicated reasons. The