Liver regeneration is accompanied by a series of profound changes in hepatocyte gene expression. Similar changes in gene expression occur when hepatocytes are placed in primary culture. In the present study, we wished to determine the in vitro patterns of gene expression for several different functional classes of hepatocyte genes, and whether the changes in gene expression depended on the extracellular matrix. We therefore examined the expression of several genes which are known to change during hepatocyte proliferation in vivo or which are known to participate in different aspects of differentiated hepatocyte function. Hepatocytes were plated on collagen Type I, a matrix which supports cellular proliferation, or on the basement membrane matrix Matrigel, which supports a non-proliferating, differentiated hepatocyte phenotype. Total cellular RNA was collected after various times in culture, and cellular mRNA levels were determined by northern blot analysis for the following: transcription factors liver regeneration factor-1 (LRF-1) and CCAAT/enhancer binding protein delta (CEBPδ), the microsomal enzyme cytochrome CYP2A3 (rat orthologue of mouse CYP2A5) and the cell surface heparan sulfate proteoglycan, syndecan-2 (syn-2). Marked and rapid increases in LRF-1 and CEBPδ expression were observed on both matrix types. However, the expression of LRF-1 decreased rapidly on both matrices at 24 h, whereas CEBPδ expression was more delayed and returned to baseline values after 48 h. Messenger RNA for CYP2A3 decreased over 24 h on both matrices, but returned to 46% of control after 48 h on Matrigel which was significantly higher than hepatocytes plated on collagen. Finally, the expression of syndecan-2 was markedly decreased when hepatocytes were cultured on collagen Type I, but remained higher for hepatocytes grown on Matrigel at 48 h. For syndecan-2 it was shown that the half-life of syndecan mRNA was 1.5 fold longer on Matrigel than on collagen for the 3.4 kb mRNA, but no differences were found for the 2.2 and 1.1 kb messenger RNAs. These studies suggest that many changes in gene expression which occur in vivo during liver regeneration also occur in vitro during the transition to culture, and that some of these changes are influenced by the matrix upon which the cells are grown. Furthermore, these studies demonstrate that matrix-dependent changes in mRNA stability contribute, at least in part, to the observed changes in gene expression.
To elucidate the possible pathomechanism of glomerulosclerosis by partial nephrectomy, male SD rats received 3/4 nephrectomy and were maintained without further treatment for 8 weeks (3/4 nephrectomized group). Another group received a sham operation consisting of laparotomy and manipulation of the renal pedicles but without destruction of renal tissue and were maintained without treatment for 8 weeks (sham-operated group). The following findings were observed in the 3/4 nephrectomized group. The levels of urinary protein elevated after 4 weeks and thereafter reached a plateau. A high blood pressure was present at week 8. Histopathologically, glomerulosclerotic lesions were characterized by capillary obsolescence, proliferation of glomerular cells (including mesangial cells), mesangial matrix increase, and macrophage influx, and associated with expression of α-smooth muscle actin (α-SMA) and desmin in the glomerular tuft (mainly mesangial cells) and platelet-derived growth factor (PDGF) receptor in the glomerular tuft. The present results suggest that the key pathologic changes for glomerulosclerosis are impaired endothelial cells of glomerular capillaries by rapidly increased blood flow into the remaining nephrons and the resultant production of growth factors such as PDGF leading to mesangial cell proliferation. Furthermore, the present expression of α-SMA and desmin in mesangial cells suggests a phenotype switch of mesangial cells responsible for the induction of glomerulosclerosis.
Early histological changes in the mucosa of the respiratory system were examined in Brown Norway (BN) and Fischer 344 (F344) rats exposed to aerosol inhalation of 1% formaldehyde (HCHO) solution of 3 or 5 days. Treatment-related clinical signs suggestive of respiratory disorders were more obviously observed in F344 rats than in BN rats. In F344 rats, mucosal changes were found in the nasal cavity and trachea. They were initiated by degeneration and/or desquamation of epithelial cells with inflammation and then followed by stratification or squamous metaplasia of epithelial cells. The incidence and extent of these lesions were increased with duration of exposure. On the other hand, similar but less severe changes were observed restricted to the nasal mucosa in BN rats. The degree of epithelial damage was consistent with that of mucosal inflammation, and squamous cell metaplasia occurred only in the portion where severe mucosal lesions preexisted. The present study confirms that BN rats are less sensitive to HCHO inhalation than F344 rats.
Renal tubulointerstitial lesions in mercuric chloride-treated Brown Norway rats were investigated focusing on the relationship between the development of lesions and kinetics of infiltrating cells and α-smooth muscle actin (SMA)-positive myofibroblasts. Fifteen rats were injected with 1 mg/kg b.w. of mercuric chloride at day 0, and 5 rats were killed at days 2, 4, and 6, respectively. Another 20 rats were injected with the same dose of mercuric chloride at days 0, 2 and 4, and 5 rats were killed at days 6, 8, 10, and 20, respectively. No additional changes were detected in glomeruli, whereas, mainly in the proximal tubules, necrosis was prominent at day 2, regenerative changes were obvious at day 4, and dilatation of the affected tubules with peritubular fibrosis developed at and after day 6. Following the renal epithelial damage, mononuclear cell infiltration developed in the renal interstitium at and after day 6, and among the infiltrating cells, ED1-positive macrophages were most predominant and CD8-positive T cells were next throughout the experimental period. They began to increases prominently at 2 days after 3 injections of mercuric chloride, i.e. at day 6. In addition, the number of α-SMA-positive myofibroblasts also increased at and after day 6, and the kinetics of ED1-positive macrophages corresponded well with that of α-SMA-positive myofibroblasts. Renal interstitial fibrosis developed along with the increase in numbers of these two cell populations. As to other infiltrating cells, although the absolute number was very small, CD45RA-positive B cells significantly increased at and after day 10. A few CD4-positive T cells and ED2-positive cells were also observed but their numbers did not show significant changes. The present results indicate that ED1-positive macrophages and α-SMA-positive myofibroblasts participate in renal fibrosis in Brown Norway rats following mercuric chloride-treatment, although the role of lymphocytes in renal fibrosis is still obscure.
Expression of glutathione-S-transferase P-form (GST-P) mRNA revealed by in situ hybridization histochemistry was investigated in enzyme altered foci and nodules during rat hepatocarcinogenesis, using diethylnitrosamine initiation followed by 2-acetylaminofluorene promotion protocol. The majority of early foci showed uniformly high levels of GST-P mRNA, however, some lesions after completion of the promotion regimen exhibited a progressive loss of expression of GST-P mRNA. Most of the continuous and strong GST-P mRNA positive lesions were also transforming growth factor-α (TGF-α)-positive lesions, a putative potent mitogen for altered hepatocytes. Moreover, the proliferating cell nuclear antigen (PCNA) index was significantly (p<0.05) greater in the GST-P mRNA-positive nodules than that in negative ones. These results suggest that stable expression of GST-P mRNA is a very useful marker for identification of developing preneoplastic lesions during rat hepatocarcinogenesis.
In this study the inhibitory effect of a high dose of estrogen on mammary carcinoma growth was investigated, especially in relation to apoptosis and mitosis, and compared this to the effects of ovariectomy. A transplantable mammary carcinoma with estrogen receptors was induced in a female Sprague-Dawley rat by multiple intragastric intubations of 7, 12-dimethylbenz(a)anthracene. The mammary carcinoma was then transplanted into female rats, and on the 21st day after the transplantation the rats were divided into three groups. Rats in two of these groups were injected with either vehicle or 1.0 mg of 17β-estradiol three times a week, and rats in the remaining group were ovariectomized. Rats were sacrificed on either the 1st, 7th, 14th, or 21st day after the initial administration or ovariectomy. The growth of transplanted mammary carcinomas was suppressed in ovariectomized rats and rats treated with a high dose of 17β-estradiol. In the 17β-estradiol treated rats, the incidences of apoptotic cells and Apop Tag positive cells were higher than those of the intact control on the 7th to 21st day after the initial administration, and the incidences of mitotic cells and 5-Bromodeoxyuridine staining positive cells were higher only on the 7th day after the initial administration. Conversely, there was no difference between incidences of apoptotic cells and Apop Tag positive cells between the ovariectomized rats and the intact control throughout the treatment period, and incidences of mitotic cells and 5-Bromodeoxyuridine staining positive cells were lower in the ovariectomized rats on the 7th to 21st day after the ovariectomy. These results indicate that the growth inhibition of the mammary carcinoma in rats treated with a high dose of 17β-estradiol was related to apoptosis. In contrast, a decrease of cell proliferation is considered to be the mechanism responsible for the inhibitory effect of ovariectomy on the growth of the mammary carcinoma.
Lectin histochemical study was done on canine mammary gland epithelial cells of 6 groups (normal, hyperplasia, adenoma, adenocarcinoma, benign mixed tumor and malignant mixed tumor). Among ten lectins used, three lectins showed an apparent alteration in the binding activity following neoplastic changes. Namely, Dolichos biflorus and Helix pomatia specific for terminal N-acetylgalactosamine residue bound more strongly to the surface of epithelial cells in adenocarcinoma and malignant mixed tumor. In addition, Ulex europaeus specific for terminal fucose residue showed stronger binding activity in mammary neoplasma irrespective of malignancy.