Immunohistochemical localization of laminin, nidogen, and type IV collagen during the early development of human liver

 There is evidence that basement membrane components control differentiation of liver sinusoids and bile ducts. These processes occur in humans in the 9th gestational week (GW). Distribution of laminin, nidogen, and type IV collagen was studied during human liver development between the 6th and the 10th GW. Laminin and nidogen lined intrahepatic microvessels in the 6th and 7th GW decreasing in quantity at the beginning of the fetal period (9th–10th GW). Type IV collagen was detected in microvessels only from the 9th GW onward. In the early periportal matrix (9th–10th GW) laminin, nidogen, and type IV collagen were diffusely distributed. At these stages, basement membrane zones of larger portal vessels and of early bile ducts were also stained for all three glycoproteins. These results show that laminin and nidogen are localized in microvessels during early human liver development and decrease in concentration at the developmental stage during which microvessels become discontinuous. In contrast, type IV collagen is not present in early microvessels but occurs when laminin and nidogen disappear. The three glycoproteins occur together only in those areas of the developing liver in which, from the 9th GW onward, the differentiation of immature liver cells into biliary epithelium takes place. Accepted: 20 August 1998     

Changes in lectin binding sites during early human liver development

 In this study we investigated whether changes in glycosylation during liver morphogenesis correlate with the early development of individual structures in the human liver. Therefore, we localized the binding of the lectins from Sambucus nigra (SNA; specific for sialic acid), Triticum vulgare (WGA; specific for N-acetylglucosamine and sialic acid), Ricinus communis (RCA I; specific for β-galactose), Lotus tetragonolobus (LTA; specific for α-fucose) and Concanavalia ensiformis (Con A; specific for α-mannose) in the human liver between the 4th and the 12th gestational week (GW). Cell membranes of early hepatocytes (5th–6th GW) showed strong staining for RCA I, which decreased noticeably from the 8th–9th GW onward. Early intrahepatic capillaries (4th–5th GW) showed reactions only for WGA and RCA I. Reactions for SNA occurred later (6th–9th GW). At this time a fine granular staining for SNA was visible at the sinusoidal sides of hepatocytes. The hepatocytes of the outer limiting plate were specifically stained by WGA, Con A, and SNA in the 9th GW and the staining remained visible in developing bile ducts up to the 12th GW. The possible biological significance of the appearance or disappearance of carbohydrate moieties during early human liver development is discussed. Accepted: 21 October 1996     

Expression of different liver cell markers during the early prenatal human development

The expression of the liver cell markers, vimentin, desmin, cytokeratins 7, 18, 19, and stem cell markers CD34 and Bcl-2 in the early stages of the human prenatal development was studied. Desmin was revealed in sinusoidal liver cells between 3.5 and 12 weeks of gestation; in mesenchymal cells of ventral mesentery and hepatoblasts it was detected at the 4–7th weeks of gestation. During the hepatic period of hemopoiesis, desmin-positive sinusoidal cells were located close to blood cells. So-called “cholangio-” cytokeratins 7 and 19 displayed different expression patterns. Cytokeratin 7 was found only in cholangiocytes, and cytokeratin 19 in hepatoblasts until 15–16 weeks of prenatal development. Mesenchymal cells of the ventral mesentery expressed cytokeratins 18 and 19 more than hepatoblasts at the 4–7th weeks of gestation. Bcl-2 was seen in the same period in most sinusoidal and mesenchymal cells of the ventral mesentery. CD34 positive cells were detected in liver sinusoids between the 4th and 9th weeks of gestation but probably they are not progenitors of hepatocytes during embryonic development. Ventral mesentery mesenchyma was negative for CD34. These results let us hypothesize that hepatocytes and cholangiocytes may arise from different embryonic sources: cholangyocytes derive only from duodenal epithelial cells, while hepatoblasts develop most likely with the participation of mesenchymal cells.     

Global gene expression profiling reveals a key role of CD44 in hepatic oval-cell reaction after 2-AAF/CCl<Subscript>4</Subscript> injury in rodents

Liver progenitors, so-called oval cells, proliferate remarkably from periportal areas after severe liver injury when hepatocyte regeneration is compromised. These cells invade far into the liver parenchyma. Molecular mechanisms underlying these behaviors of oval cells remain poorly understood. In this study, we treated rats with 2-acetylaminofluorene/carbon tetrachloride to induce hepatic oval cells. By expression microarray analysis, we investigated global gene expression profiles in liver tissue, with an emphasis on adhesion molecules, extracellular matrix proteins, matrix metalloproteinases (MMPs), growth factors/cytokines, and receptors that might contribute to the distinct behaviors of oval cells. Genes upregulated at least twofold were selected. We then performed immunostaining to verify the microarray results and identified expression of MMP-7 and CD44 in oval cells. Staining of cytokeratin (CK)-19, an oval-cell marker, was similar between oval cells located next to periportal areas and those located far within the parenchyma. In contrast, CD44 staining was more intense in the parenchyma than in periportal areas, suggesting a role of CD44 in oval-cell invasion. Moreover, newly differentiated CK-19⁺ hepatocytes within foci did not show CD44 staining, suggesting that CD44 is related to the undifferentiated oval-cell phenotype. We then investigated oval-cell reactivity in CD44-deficient mice fed an oval cell-inducing diet of 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Results showed significantly reduced oval-cell reactivity in CD44-deficient mice. Thus, oval cells express MMP-7 and CD44, and CD44 appears to play critical roles in the proliferation, invasion, and differentiation of hepatic oval cells in rodents.     

Cell sources of liver development

The work is devoted to consequent expression of different cell types' protein markers such as vimentin, desmin, cytokeratins 7, 18, 19, stem cell markers CD34 and Bcl-2 at early stages of human prenatal development. Desmin was revealed in sinusoidal liver cells on 3.5-12 weeks of gestation, in mesenchymal cells of ventral mesentery and hepatoblasts on the 4-7 accordingly. During hepatic period of blood formation such desmin positive sinusoidal cells were found to be located close to blood cells. So called "cholangio-" cytokeratins 7 and 19 showed different expression, the first one was found only in cholangiocytes, while cytokeratin 19 existed in hepatoblasts as well until week 15-16 of prenatal development. Mesenchymal cells of ventral mesentery are positive for cytokeratins 18 and 19 even brighter than hepatoblasts in the 4-7 weeks of gestation. Bcl-2 expression was seen in the same periods in most sinusoidal and mesenchymal cells of ventral mesentery. CD34 positive cells are strongly depicted in liver sinusoids from 4th until 9th weeks of gestation, but probably they are not a source of hepatocytes' development in embryonic ontogenesis. Ventral mesentery mesenchyme was negative for this very marker. These results let us suppose that hepatocytes and cholangiocytes may develop from quite different embryonic sources: cholangyocytes grow exceptionally from duodenum epithelial cells, while there is a strong possibility that hepatoblasts formation occurs with participation of mesenchymal cells.     

cAMP-mediated upregulation of gelatinases in primary cultures of isolated rat hepatocytes

Matrix metalloproteinases (MMPs) play a major role in tissue remodelling and repair in pathophysiological conditions, such as liver fibrosis and regeneration. Regulation of the MMPs produced by liver cells is important in maintaining cell-matrix ratio in liver, which is a major target site for hormones that mediate their intracellular effects through cAMP. The possibility of cAMP affecting the activity of MMPs and their endogenous inhibitors, tissue inhibitor of MMPs (TIMPs) was studied using isolated rat hepatocytes in culture. Zymographic analysis showed that treatment with hormones like epinephrine, thyroxine and dexamethasone and Bt2 cAMP increased 92 kDa MMP-9 activity. Bt2 cAMP caused upregulation of MMP-9 in a dose-dependent manner. The effect of hormones was less on MMP-2. ELISA using specific antibodies showed increase in levels of MMP-9 and TIMP-1 protein. Kinetic analysis of production of MMPs and TIMPs showed that the response to Bt2 cAMP was a delayed one, indicating its effect on de novo protein synthesis. These results suggest the possibility of cAMP dependent regulation of MMP-9 in the hepatocytes.     

Early effects in chemical-induced rat liver carcinogenesis: an immunohistochemical study following exposure to 0.04% AAF

To investigate effects that distinguish AAF from incomplete carcinogens, the rate of cell death (apoptosis) and cell proliferation was studied at early stages of AAF induced rat liver carcinogenesis. Male Wistar rats were fed 0.04% AAF in the diet for 2, 6 and 16 weeks and immunohistochemical markers were measured in the liver. The formation of initiated cells and preneoplastic foci was followed by staining for GST-P (glutathione-S-transferase). GST-P-positive foci were present from 6 weeks on. Apoptosis was increased in the periportal area and in preneoplastic foci at all time points. Cell proliferation was enhanced in the periportal area in oval cells and in bile duct-like cells particularly at 2 and 6 weeks and mainly in GST-P positive foci at 16 weeks. Notably, more cells always proliferated than were eliminated. Other apoptosis-related markers like p53 and FAS/Apo-1 could not be demonstrated in either normal hepatocytes, preneoplastic foci or in hepatocytes from treated animals. Scattered bcl-2 positive cells were present in livers at 16 weeks of treatment. The two cell growth and differentiation related proto-oncogenes c-FOS and c-JUN were increased in all treated animals at early stages. If feeding was stopped after 6 weeks, livers did not recover significantly within the following 10 weeks. The results support the complex effects of AAF in rat liver carcinogenesis. Chronic toxicity locally impairs the balance between cell proliferation and cell death and induces morphological alterations that promote the growth of initiated cells.     

Maintenance of periportal and pericentral oxygen tensions in primary rat hepatocyte cultures: influence on cellular DNA and protein content monitored by flow cytometry

Primary hepatocytes were cultured at oxygen tensions similar to those reported to be present in periportal (13% O2) and pericentral (4% O2) regions of the liver lobules. Cellular DNA and protein content of individual hepatocytes were determined simultaneously by two-parameter (DNA/protein) flow cytometry after 1, 4, and 7 days in culture. pO2 tensions monitored on line in conventional plastic culture dishes revealed that the depletion of the pO2 in the culture medium depended on the number of hepatocytes plated. When cultured as monolayer after 4-7 days at periportal (13% O2) and more pronounced at pericentral oxygen concentration (4% O2), up to 90% of the hepatocytes showed degenerated nuclei but normal protein content. By using culture dishes with teflon membrane bottoms the oxygen tension in the culture medium was accurately maintained by the incubator atmosphere. At pericentral oxygen tension the fraction of 2N cells increased by about 20%. That of the 4N cell was not affected, and the contribution of 8N hepatocytes dropped to 70% compared to cultures at periportal oxygen tension. Concomitantly, in the 4% O2 hepatocyte cultures the protein content of the 2N and the 4N cells was better preserved and increased by up to 10%. These results suggest that in vitro at pericentral oxygen conditions (4% O2) ageing of hepatocytes is delayed, regenerating processes are better maintained, and, furthermore, freshly isolated 4N hepatocytes have the potency to adapt their metabolism in vitro to periportal as well as to perivenous oxygen tensions.     

Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes

Two antigens - A6 and G7 - shared by mouse biliary epithelial and oval cells were revealed by monoclonal antibodies raised in rat immunized with oval-cell-enriched liver fraction. Oval cells were induced in CBA or F1 (CBA x C57BL6) mice by a combination of a single injection of the alkylating drug Dipin with partial hepatectomy. In normal liver A6 antigen was localized, using light and electron microscopy, in biliary epithelial cells of all ducts including Hering canals. Some bile ductal and Hering cells were A6-negative. Occasionally, A6 antigen was present in single hepatocytes forming the periportal ends of hepatic cords. In preneoplastic and tumorous liver A6 antigen was present in bile ductal and oval cells and in a fraction of newly formed hepatocytes and tumor cells. G7 antigen was revealed in normal, precancerous and tumorous liver in biliary epithelial and oval cells but not in hepatocytes. A6 and G7 antigens were not liver-specific: they were expressed in various normal organs and tissues, especially in epithelia. In studies of mouse liver lineages A6 antigen can be used as a common marker of biliary epithelial and oval cells and hepatocytes at certain stages of differentiation. G7 antigen is a marker of oval and biliary epithelial cells. There was a striking similarity in A6 antigen localization to that of human blood group antigens in normal liver and liver tumors. A6 antigen may thus provide a useful tool for the study of neoexpression of human blood group antigens in liver tumors.     

Characterization of two distinct liver progenitor cell subpopulations of hematopoietic and hepatic origins

Despite extensive studies, the hematopoietic versus hepatic origin of liver progenitor oval cells remains controversial. The aim of this study was to determine the origin of such cells after liver injury and to establish an oval cell line. Rat liver injury was induced by subcutaneous insertion of 2-AAF pellets for 7 days with subsequent injection of CCl(4). Livers were removed 9 to 13 days post-CCl(4) treatment. Immunohistochemistry was performed using anti-c-kit, OV6, Thy1, CK19, AFP, vWF and Rab3b. Isolated non-parenchymal cells were grown on mouse embryonic fibroblast, and their gene expression profile was characterized by RT-PCR. We identified a subpopulation of OV6/CK19/Rab3b-expressing cells that was activated in the periportal region of traumatized livers. We also characterized a second subpopulation that expressed the HSCs marker c-kit but not Thy1. Although we successfully isolated both cell types, OV6/CK19/Rab3b(+) cells fail to propagate while c-kit(+)-HSCs appeared to proliferate for up to 7 weeks. Cells formed clusters which expressed c-kit, Thy1 and albumin. Our results indicate that a bona fide oval progenitor cell population resides within the liver and is distinct from c-kit(+)-HSCs. Oval cells require the hepatic niche to proliferate, while cells mobilized from the circulation proliferate and transdifferentiate into hepatocytes without evidence of cell fusion.     

TGF-beta3-induced palatogenesis requires matrix metalloproteinases

Cleft lip and palate syndromes are among the most common congenital malformations in humans. Mammalian palatogenesis is a complex process involving highly regulated interactions between epithelial and mesenchymal cells of the palate to permit correct positioning of the palatal shelves, the remodeling of the extracellular matrix (ECM), and subsequent fusion of the palatal shelves. Here we show that several matrix metalloproteinases (MMPs), including a cell membrane-associated MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) were highly expressed by the medial edge epithelium (MEE). MMP-13 was expressed both in MEE and in adjacent mesenchyme, whereas gelatinase A (MMP-2) was expressed by mesenchymal cells neighboring the MEE. Transforming growth factor (TGF)-beta3-deficient mice, which suffer from clefting of the secondary palate, showed complete absence of TIMP-2 in the midline and expressed significantly lower levels of MMP-13 and slightly reduced levels of MMP-2. In concordance with these findings, MMP-13 expression was strongly induced by TGF-beta3 in palatal fibroblasts. Finally, palatal shelves from prefusion wild-type mouse embryos cultured in the presence of a synthetic inhibitor of MMPs or excess of TIMP-2 failed to fuse and MEE cells did not transdifferentiate, phenocopying the defect of the TGF-beta3-deficient mice. Our observations indicate for the first time that the proteolytic degradation of the ECM by MMPs is a necessary step for palatal fusion.     

Exogenous prostaglandin E2 inhibits TPA induced matrix metalloproteinase-9 production in MCF-7 cells

Elevated levels of prostaglandin E2 (PGE2) have been reported in many high metastatic human breast cancers, but no relationship between exogenous PGE2 activity, expression of matrix metalloproteinases (MMPs) and metastasis in human tumor cells has been reported. The poorly invasive human breast cancer cell line MCF-7 was cultured for 24h in the presence of both phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 nM) and PGE2 (1 microM) and the activity of MMP-9, one of the MMPs involved in metastasis, was measured, in growth medium by gelatin substrate zymography. TPA induced a strong production of MMP-9 while exogenous PGE2 had no effect on the basal MMP-9 level, but inhibited the TPA induced enzyme expression and matrigel invasiveness. We showed that MCF-7 cells expressed EP2, EP3 and EP4 receptors for PGE2 and that its action was probably mediated by EP4 receptor and adenylyl cyclase activation while cAMP dependent PKA was not involved in the process of inhibition of MMP-9 production. These findings suggest a possible inhibitory role for exogenous PGE2 in the metastatic process development.     

Effect of Thrombin on Human Amnion Mesenchymal Cells,Mouse Fetal Membranes,and Preterm Birth

Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE₂ in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE₂ content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion.     

Roles of MMP/TIMP in regulating matrix swelling and cell migration during chick corneal development

Tissue remodeling is central to embryonic development. Here, we used immunohistochemistry, Western blotting, and RT-PCR analysis to investigate the roles of matrix metalloproteinases (MMPs) and the related "a disintegrin and metalloproteinase" (ADAM) family proteinases in chick corneal development. While MMP-13 was expressed in developing chick corneas from embryonic day (ED) 5 to ED 10, its inhibitor, tissue inhibitors of metalloproteinase-1 (TIMP-1), was expressed from ED 18 to 2 days post-hatching (P2). Early MMP-13 activity may be associated with degradation of type IX collagen from the primary stroma, which loosens the collagen fibrils and facilitates neural crest (NC) cell migration. The membrane-bound and secreted forms of ADAM10 were both detected throughout corneal development, and active ADAM10 formed a cleavage complex with CD44v6, a CD44 splice variant that is a major cell surface adhesion molecule for hyaluronic acid (HA) and has been implicated in cell migration. Both CD44v6 and its ectodomain cleavage products were detected from ED 5 to ED 14, and a broad-spectrum MMP inhibitor blocked ectodomain cleavage in cultured stromal cells. These findings suggest that ADAM10 mediates CD44v6 cleavage in the developing cornea, facilitating NC cell-derived mesenchymal cell migration. Finally, we identified high levels of active membrane-type 3-MMP (MT3-MMP) in developing corneas at ED 7, ED 14, and ED 18. MT3-MMP takes part in MMP-2 activation and possibly also CD44v6 shedding, suggesting that this pathway may be involved in cell migration. These findings collectively show for the first time that multiple MMPs, ADAMs, and TIMPs appear to functionally interact during corneal development.     

Hepatocyte Produced Matrix Metalloproteinases Are Regulated by CD147 in Liver Fibrogenesis

Background

The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC.

Methods

Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl₄ induced liver injury using ãCD147 antibody intervention.

Results

In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl₄ treated mice compared to controls.

Conclusion

We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by future anti-fibrogenic agents.     

胎肝中肝干细胞的免疫组织化学研究

目的采用免疫组织化学方法显示不同时期人胚胎肝脏的干细胞,分析肝干细胞的形态与分布特点及发育过程中干细胞在肝脏中的迁徙,探讨肝脏的发生发育及肝内干细胞的来源。方法不同发育时期胎儿肝脏,取材、固定、制成石蜡切片,ABC法检测肝干细胞特异性的表面标记物CD34、CK19、C-11和OV6。结果胎肝内汇管区周边界板处有卵圆样细胞表达CD34、C-11、CK19和OV6,阳性细胞紧密排列成管,呈鞘样包绕着早期汇管区,部分包绕着初级汇管区,随着次级汇管区的成熟,卵圆样干细胞逐渐局限于赫令氏管周围;此外,胚胎发育的不同阶段均可见CD34、OV6阳性的单核样细胞分散在肝索、肝血窦之内,多见于汇管区的问充质组织之内,肝血管内鲜见。结论胚胎发育早期汇管区周边界板处含有丰富的干细胞,可能是肝脏发育的起点,这些干细胞逐渐分化为胆管上皮样细胞,然后分化为肝细胞和胆管上皮细胞;造血干细胞是肝内的另一干细胞来源,造血干细胞在肝内受到诱导作用分化为小部分的肝实质细胞。     

Secretion and gene expression of metalloproteinases and gene expression of their inhibitors in porcine corpora lutea at different stages of the luteal phase

We hypothesize that spontaneous regression of corpora lutea (CL) involves short-lasting restructure of luteal tissue with an activation of matrix metalloproteinases (MMPs) and their respective inhibitors (tissue inhibitors of metalloproteinase, TIMPs). This was tested by determining the gene expression of MMP-1, MMP-2, and MMP-9 and respective TIMP-1 and TIMP-2 in luteal tissue from sows at the early, midluteal, and late luteal phase (Days 6-8, Days 9-11, and Days 13-15 of estrous cycle). Gene expression of the three MMPs was low in early, slightly higher in midluteal, and significantly elevated (P < 0.05) in regressing CL. An inverse pattern was found for gene expression of TIMP-1 and TIMP-2. Under culture conditions, the release of MMPs was determined from steroidogenic large luteal cells (LLC). LLC harvested from regressing CL released significantly (P < 0.05) more active MMPs than cells obtained from CL at the early luteal phase. As luteolysis can be induced by prostaglandin F(2alpha) (PGF(2alpha)) and tumor necrosis factor alpha (TNF), we studied their effects on LLC under culture conditions. Treatment of cells with PGF(2alpha) or TNF (10(-7) M or 3 x 10(-9) M, respectively) induced a significantly higher release of MMPs, and gene expression was also significantly stimulated in comparison to that in untreated LLC. The gene expression of TIMPs remained unaffected by either treatment. It is concluded that at the beginning of luteolysis, MMPs are expressed and released in high amounts and that this is essential for the structural regression of the CL.