Effects of transforming growth factor beta on corneal epithelial and stromal cell function in a rat wound healing model after excimer laser keratectomy

BACKGROUND: Transforming growth factor beta (TGF-beta) regulates extracellular matrix deposition, cell proliferation, and migration, and is expressed in cornea. TGF-beta is thought to be involved in the corneal wound healing process. METHODS: The central corneal area (3 mm in diameter) of Lewis rats was ablated using PTK mode excimer laser and the wound healing process was observed at 12 and 24 h and 2, 5, 10, and 30 days after treatment. The expression of TGF-beta 1, -beta 2 and -beta 3, TGF-beta type I and type II receptors, alpha 3, alpha 5, beta 4 integrin subunits, laminin and fibronectin was studied immunohistochemically. Antibody neutralizing TGF-beta 1, -beta 2 and -beta 3 was administered intraperitoneally, 50 micrograms daily, for 5 days after the laser treatment to investigate the effects of TGF-beta function blockade. RESULTS: At the leading edge of the regenerating epithelium, no TGF-beta type I and type II receptors and beta 4 integrin subunits were expressed after 24 h. Regenerating epithelium covered the ablated area after 2 days. An abnormal fibrotic layer was formed in the subepithelial area. This layer contained round-shaped cells in the stroma in the early stage (2-5 days after laser ablation) and spindle-shaped fibroblast-like keratocytes after 10 days. Laminin and fibronectin expression increased in the fibrotic layer. The increased stromal cells expressed TGF-beta isoforms and TGF-beta receptors. Neutralizing TGF-beta inhibited the stromal cell increase in the laser ablated area after 5 days. CONCLUSION: TGF-beta may be involved in epithelial cell migration and stromal cell reaction during the corneal wound healing process after excimer laser ablation in rat models.     

Cooperation of Ito cells and hepatocytes in the deposition of an extracellular matrix in vitro

Cellular and molecular mechanisms involved in the deposition of extracellular matrix components in both normal and fibrotic liver are still poorly understood. We have investigated the influence of cooperation between Ito cells and hepatocytes in matrix deposition in vitro. Immunoprecipitation of radiolabeled proteins from media of 5-day-old Ito cell primary cultures showed that these cells secreted high levels of the major basement membrane components, ie, collagen IV, laminin, and entactin/nidogen. By immunocytochemistry, precursors of basement membrane components were found intracellularly, but only scarce deposits were seen around the cells. When hepatocytes were added to 2-day-old Ito cell primary cultures, they established close contacts with Ito cells in less than 24 hours and expressed ZO-1, a tight junction-associated protein not detectable in standard hepatocyte culture. Cytochemistry analysis revealed an abundant extracellular matrix deposited over hepatocyte cords and between hepatocytes and Ito cells. Immunocytochemistry studies showed that this matrix contained laminin, fibronectin, and collagens proIII and IV. These data indicate that a high level of matrix protein synthesis by liver cells in vitro is not sufficient to induce extracellular matrix deposition, and that cell-cell interactions are strongly involved in this process. Hepatocyte/Ito cell co-culture, which may reflect the actual situation in vivo, represents a useful tool for studying liver fibrogenesis.     

Laminin localization in enterocytic basement membrane of rat small bowel grafts. A light and electron microscopic study

In vitro laminins stimulate numerous biological effects, such as cell migration, proliferation, attachment and differentiation. In vitro laminins influence immunocompetent cells and in vivo possibly play an important role in graft rejection. To establish how laminins could be involved in the regulation of acute rejection of small bowel allografts (with and without immunosuppression), we investigated laminin distribution in rat small bowel allografts four days after transplantation, i.e., before the onset of histological signs of rejection, using antibodies against alpha1, beta1, gamma1 chain of laminin-1. In immunosuppressed allografts, the ultrastructure of the enterocytic basement membrane appeared normal, but no laminin staining was seen in this membrane, although basement membranes of intramural blood vessels and muscle cells were normally stained. In non-operated immunosuppressed rats, laminin staining was clearly reduced in the enterocytic basement membrane, demonstrating that cyclosporin A is able to affect this membrane. Since only rats in which laminin is altered survive, this laminin alteration in the enterocytic basement membrane presumably plays an important role in overcoming the acute rejection.     

Substratum-dependent and region-specific control of attachment and proliferation of gastrointestinal epithelial cells in primary serum-free culture

A system for the primary serum-free culture of fetal rat gastrointestinal epithelial cells was used to examine the role of the extracellular matrix (ECM) in the attachment and proliferation of these epithelial cells. Forestomach epithelial cells (FSEC) were able to attach to and proliferate on plastic dishes without a substratum, while glandular stomach epithelial cells (GSEC) and duodenal epithelial cells (DEC) were unable to do so. The presence of a substratum promoted the attachment and proliferation of these epithelial cells. The effects of various components of the ECM differed depending on the type of cell. FSEC attached most efficiently to a substratum of fibronectin, while GSEC did so to laminin. DEC attached more efficiently to type I collagen and fibronectin than to any other substratum. FSEC proliferated most rapidly on laminin, while GSEC and DEC did so on collagen gels. These substrata induced the most efficient attachment and proliferation of FSEC, and they were effective in promoting the attachment and proliferation of GSEC and DEC in decreasing order of efficiency, indicating the existence of a head-to-tail gradient in the response of epithelial cells to substrata. The expression of c-myc mRNA in these cells differed depending upon the substratum on which they were cultured and the mRNA level was well correlated with the extent of the cell proliferation, indicating that the cell proliferation is mediated by c-myc gene expression, which is regulated by cell-ECM interactions. The results of the present study demonstrate that proliferation of gastrointestinal epithelial cells is regulated region-specifically not only by soluble factors but also by insoluble components of the ECM.     

Interferon gamma decreases hepatic stellate cell activation and extracellular matrix deposition in rat liver fibrosis

Interferon gamma (IFN-gamma) inhibits in vitro the activation of hepatic stellate cells (HSC), the primary extracellular matrix-producing cells in liver fibrosis. This study was undertaken to determine in vivo the effect of IFN-gamma in the rat model of liver fibrosis induced by dimethylnitrosamine (DMN), where HSC activation represents an early response to cell injury. Rats were killed after 1 or 3 weeks of treatment with DMN, IFN-gamma, DMN + IFN-gamma, or saline. Immunohistochemistry was used to identify proliferating (desmin-positive/bromodeoxyuridine (BrdU)-positive cells) and activated (alpha-smooth-muscle actin [alpha-SMA]-positive cells) HSCs. Collagen deposition was determined colorimetrically and by morphometry. The parenchymal extension of desmin- and actin-positive cells and of fibrotic tissue was measured by point-counting technique and expressed as a percentage of area. Western blot was used to determine laminin and fibronectin accumulation. The levels of messenger RNA (mRNA) for procollagen type I, fibronectin, and laminin were evaluated by Northern blot. No differences were observed in rats treated with either saline or IFN-gamma alone. IFN-gamma reduced HSC activation induced by liver injury, as shown by the decreased number of proliferating HSC and the reduction of parenchymal area occupied by alpha-SMA-positive cells observed in DMN + IFN-gamma-treated animals compared with the DMN group. This was associated with reduced collagen, laminin, and fibronectin accumulation and lower levels of mRNA for procollagen type I, fibronectin, and laminin in the DMN + IFN-gamma group. Thus, this study indicates that IFN-gamma reduces extracellular matrix deposition in vivo by inhibition of HSC activation.     

The reversed-flow medio-distal fasciocutaneous island thigh flap: anatomic basis and clinical applications

To investigate changes in retinal pigment epithelial (RPE) cells during wound healing, we evaluated the deposition of newly synthesized extracellular matrix (ECM) over time during wound healing in rat RPE cultures. We also estimated the effect of growth factors on the healing rate and ECM synthesis. After preparing rat RPE cell sheet cultures, we made round 1-mm defects in the cultures. Fibronectin, laminin, and collagen IV synthesis were evaluated with immunocytochemistry every 12 hours after wounding. S-phase cell distribution was analyzed every 12 hours by 5-bromodeoxyuridine uptake. We added either platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor- beta2 (TGF-beta2) to cultures at concentrations of 1, 10, and 100 ng/mL and immunocytochemically analyzed the effects on ECM and estimated the rate of wound closure. Although approximately 50% closure was achieved 24 hours after wounding, fibronectin deposits first appeared at that time. Laminin and collagen IV were first detected at 36 hours and fibronectin staining had extended toward the wound center. S-phase cells were distributed in concentric rings that moved centripetally over time and corresponded to the leading edge of the area stained with anti-ECM antibodies. TGF-beta2 enhanced ECM deposition, but EGF and PDGF did not. TGF-beta2 decreased the healing rate in a dose-dependent manner, whereas PDGF promoted wound closure. EGF enhanced closure at the highest concentration only. In summary, wound healing in RPE may be initiated when cells at the wound edge slide or migrate toward the wound center, which is followed by cell proliferation and then ECM synthesis. ECM components may be produced in a specific sequence during healing. TGF-beta2 may promote RPE cell differentiation, and PDGF may enhance proliferation during wound healing of the RPE.     

The effect of osteotomy on bowing and height in children with X-linked hypophosphatemia

Extracellular matrices (ECM) are utilized for obtaining better cell attachment to polymer surfaces in cell cultures. To establish beneficial bioartificial renal tubules, tubular epithelial cells and ECM were investigated in this study. MDCK cells and KU-2 cells were seeded onto 96 well plates which had been precoated with collagen types I and IV, laminin, and fibronectin. The culture media were removed and replaced with new ones at 15, 30, 60, 90, 120, and 150 min and 24 h after start time to evaluate the incubation time effects. The degrees of cell attachment onto ECM were measured by MTT assay. In the MDCK cell culture, better cell attachment was observed between 60 min and 24 h after incubation start time with the use of laminin at a concentration of 5 microg/ml, 60 min and more after incubation start time with the use of fibronectin at the concentrations of 1 and 4 microg/ml, or 30 min and more after incubation start time with the use of fibronectin at the concentrations of 16 and 32 microg/ml. On the other hand, in the KU-2 cell culture, better cell attachment was observed between 15 and 60 min after the incubation start time or 24 h after the incubation start time with the use of laminin at a concentration of 40 microg/ml. These data suggest that various cells possibly each have a most suitable ECM kind, best concentration, and best incubation time.     

Neonatal estrogen stimulates proliferation of periductal fibroblasts and alters the extracellular matrix composition in the rat prostate

The purpose of this study was to examine whether changes in extracellular matrix (ECM) molecules are associated with the growth inhibition and differentiation defects of the prostate gland following neonatal exposure to estradiol. Using immunocytochemistry (ICC), laminin and collagen IV were localized to the basement membrane (BM) as well to the basal lamina of the periductal smooth muscle of the control developing prostates. In contrast, fibronectin and collagen III were localized throughout the stromal ECM. Exposure to neonatal estrogen altered the staining profile for specific ECM molecules. In the estrogenized rats, a thick layer of cells negative for laminin and collagen IV was observed adjacent to the BM. Electron microscopy and ICC for alpha-actin, fibronectin, and vimentin identified this multicellular layer of periductal cells as differentiated fibroblasts. Peripheral to these fibroblasts, actin-positive smooth muscle formed a second layer of periductal stromal cells. PCNA labeling showed that estrogen exposure increased the fibroblast proliferation. Because many periductal fibroblasts were positive for estrogen receptor alpha (ER alpha) in estrogenized rats, a direct effect of estradiol on their proliferation is suggested. Gelatinolytic gels revealed that estrogen exposure did not alter the activity of matrix metalloproteinases associated with tissue remodeling during prostate morphogenesis. However, the periductal fibroblast layer in estrogenized prostates was devoid of urokinase- and tissue-plasminogen activator, which may potentially alter the localized proteolysis involved in matrix remodeling. It is proposed that proliferation of a multicellular layer of periductal fibroblasts in estrogenized prostates results in a physical barrier that constrains branching morphogenesis and blocks paracrine communications between smooth muscle and epithelial cells which normally regulate differentiation.     

Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacl-galR family of regulatory genes

The rat treated with bile duct ligation (BDL) and furan is a unique animal model of massive bile ductular hyperplasia in which normal liver parenchyma is largely replaced with well-differentiated proliferated bile ductules. We have now developed a simple cell isolation procedure to obtain and culture viable bile ductular epithelial cells in high numbers and with a high degree of purity from the livers of BDL/furan-treated rats. Primary monolayer cell cultures were readily established when the isolated bile ductular epithelial cells were cultured in plastic tissue culture wells coated with rat tail tendon type I collagen plus bovine plasma fibronectin. Under these conditions, epidermal growth factor (EGF) was mitogenic for the cultured cells, and they retained phenotypic features typical of hyperplastic bile ductular epithelium but did not show evidence of ductal morphogenesis in vitro. In contrast, when the isolated bile ductular cells were cultured for 7 to 16 days in the presence of 25 ng EGF/mL and 10% fetal bovine serum on type I collagen gels, they formed into branching ductal structures whose ultrastructural features very closely resembled those of polarized hyperplastic bile ductules/ducts in vivo. Histological preparations of these gel cultures further showed that numerous ductal structures with defined lumens were present. Phenotypically, these ductal structures were completely surrounded by a thickened basement membrane that was strongly immunoreactive for laminin. Like their in vivo biliary cell counterparts, the epithelial cells comprising these ductal structures in culture also exhibited strong immunocytochemical staining reactions for cytokeratins 8 and 19, for glutathione S-transferase pi 7, and for luminal gamma-glutamyl transpeptidase, but they did not express immunoreactive albumin or alpha-fetoprotein. Occasional epithelial cells of the ductal structures, when examined in 10-day-old primary gel culture, showed strong nuclear staining for incorporated 5-bromo-2'deoxyuridine, indicating active cell proliferation. Our results support the development of a novel biliary epithelial cell culture model that has the potential of serving as a powerful tool for investigation of factors that regulate hyperplastic bile ductular morphogenesis, cell proliferation, and polarized cell functions in a structural form in vitro that mimics that of hyperplastic bile ductules induced in vivo.     

PC12 cell aggregation and neurite growth in gels of collagen, laminin and fibronectin

PC12 cells form aggregates when suspended within three-dimensional, self-assembled, type I collagen gels; these aggregates increase in size over time. In addition, when the cells are cultured in the presence of nerve growth factor, they express neurites, which extend through the three-dimensional matrix. In this report, the roles of fibronectin, laminin and nerve growth factor in PC12 cell aggregation and neurite growth following suspension in collagen matrices were evaluated. Single cells and small clusters of cells were suspended in collagen gels; the kinetics of aggregation were determined by measurement of the projected area of each aggregate, and neurite lengths were determined by measurement of end-to-end distance. Fibronectin and laminin inhibited the aggregation of PC12 cells at 50 micrograms/ml, and fibronectin, but not laminin, inhibited the growth of neurites at 100 micrograms/ml. In the absence of serum, the aggregation of cells cultured with nerve growth factor was almost completely inhibited, but the average neurite length was unaffected. In the presence of nerve growth factor, the extent of cell aggregation could not be explained simply by an increase in cell number, suggesting the presence of two separate mechanisms for aggregate growth: one dependent on cell motility and another dependent on cell proliferation.     

Liver carcinogenesis associated with feeding of ethionine in a choline-free diet: evidence against a role of oval cells in the emergence of hepatocellular carcinoma

In an attempt to clarify the role of oval cells in the emergence of hepatocellular carcinoma, we fed rats a choline-free diet containing 0%, 0.05% or 0.1% ethionine. The incidence and nature of premalignant and malignant hepatic lesions were then related to the degree of oval cell proliferation. Intake of choline-free diet alone for up to 12 mo was associated with minimal oval cell proliferation; cholangiofibrosis, hepatocellular nodules and hepatocellular carcinoma were observed in 55%, 23% and 14% of the animals, respectively. When rats were given the choline-free diet with 0.05% ethionine, proliferation of oval cells was more pronounced; after a 6- to 12 mo feeding period, cholangiofibrosis (57%) was again observed. However, hepatocellular nodules (91%) and hepatocellular carcinoma (74%) were the most common lesions seen with this feeding regimen. Finally, rats fed the choline-free diet with 0.1% ethionine had massive oval cell proliferation and progressive loss of parenchymal liver tissue. Most of these animals died before they had consumed the choline-free diet with 0.1% ethionine for 12 mo. Rats in this group (96%) exhibited large and numerous cholangiofibrotic lesions, but hepatocellular nodules and carcinoma were not detected. In all animals of each experimental group, hyperplastic bile duct cells in areas of cholangiofibrosis and oval cells were positive for cytokeratin 19, an intermediate filament protein present only in bile duct cells in normal liver. Hepatocellular nodules and hepatocellular carcinoma were invariably negative for cytokeratin 19. We interpret these findings to suggest that oval cells are not involved in the histogenesis of hepatocellular carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of extracellular matrix constituents on the attachment of human oral epithelial cells at the titanium surface

This in vitro study attempts to delineate the role of extracellular matrix (ECM) constituents at the epithelial tissue-implant interface. To know which ECM constituents have a beneficial influence on the behavior of epithelial cells, the attachment, proliferation, morphologic pattern, and differentiation or cytoskeletal organization of human oral epithelial cells on ECM-coated (type IV collagen, fibronectin, type I collagen, laminin, and vitronectin) and noncoated titanium surface have been evaluated and compared. In each experiment comparing commercially pure titanium and oxygen plasma-cleaned titanium, the same ECM constituents were used. In this study, type IV collagen could provide an excellent substratum for epithelial cell attachment on titanium surface, but vitronectin-coated titanium revealed lower effectiveness for attachment of epithelial cells than noncoated titanium. These results suggested that type IV collagen could be used as a means for obtaining good epithelial seal, whereas vitronectin could be used to restrain the attachment of epithelium to dental implants.     

Effects of the carcinogen, acrylonitrile, on forestomach cell proliferation and apoptosis in the rat: comparison with methacrylonitrile

Acrylonitrile (AN) and methacrylonitrile (MAN) are two major industrial nitriles used in the production of plastics and acrylic fibers. Whereas AN is a potent acute toxin and carcinogenic in rats, little is known regarding MAN. Current work is part of an overall effort designed to assess the potential toxicity/carcinogenicity of MAN. The present study compares the ability of the two chemicals to induce epithelial proliferation and apoptosis in the forestomach (FS; a target of AN carcinogenicity), liver and glandular stomach (non-targets of AN carcinogenicity) of male F344 rats. AN was administered to rats daily, by gavage, for 6 weeks, at 0.43 and 0.22 mmol/kg. MAN was administered at 0.87 and 0.43 mmol/kg. Both AN and MAN induced a dose-dependent increase in epithelial cell proliferation in the FS of male F344 rats as determined by bromodeoxyuridine (BrdU) incorporation into DNA. In contrast, AN, but not MAN caused a dose-dependent increase in the thickness of the forestomach squamous mucosa. This increased thickness (hyperplasia) was reflected by an increase in the number of total epithelial cells per unit length of mucosa. At doses of AN and MAN which induced a 2.3-fold increase in BrdU incorporation, apoptosis was 5- and 18-fold greater than controls, respectively. Although both MAN and AN caused a similar increase in cell proliferation, the relatively more prominent increase in the apoptotic index of the squamous epithelium of rats exposed to MAN may explain the lack of a detectable increase in the thickness of the mucosa compared to that seen with AN. The disruption of the balance between FS mucosal cell proliferation and apoptosis in favor of a net increase in the number of FS epithelial cells per unit length may contribute to the carcinogenicity of AN. In conclusion, present work demonstrated that AN selectively induced a net enhancement in FS cell proliferation, a site of its carcinogenicity. On the other hand, MAN-induced FS cell proliferation was associated with a parallel increase in apoptosis. The relatively greater increase in apoptosis by MAN may have compensated for the increase in FS mucosal cell proliferation and the lack of observable change in the FS thickness.     

Effect of heparin on mesangial cell growth and gene expression of matrix proteins

BACKGROUND: Mesangial cell (MC) proliferation and matrix expansion are characteristics of many glomerulopathies. Heparin has been shown to inhibit MC proliferation in vitro and mitigate cell proliferation, matrix expansion, proteinuria, renal insufficiency, and hypertension in experimental glomerulonephritis and subtotal renal ablation. We examined the effect of standard heparin on MC proliferation and matrix protein expression in vitro which necessarily excludes the confounding influences of haemodynamic, inflammatory, haemostatic, and various other processes that are present in vivo. METHODS: Gene expression and release of fibronectin (FN), collagen IV and laminin by cultured rat MC were tested in the presence and absence of heparin. In addition the effect of transforming growth factor-beta1 (TGF-beta1) on the gene expression of those matrix proteins was assessed. RESULTS: Within a 3-1000 microg/ml concentration range, heparin inhibited gene expression and release of FN by 10% fetal calf serum (FCS)-stimulated MC in a concentration-dependent manner. At concentrations of 300 and 1000 microg/ml, heparin inhibited fibronectin mRNA levels in TGF-beta1 (6 ng/ml) stimulated cells. However, heparin had no effect on gene expression or release of collagen IV or laminin under these conditions. Heparin markedly inhibited 10% FCS-stimulated MC proliferation in a concentration-dependent manner. CONCLUSIONS: Heparin inhibited MC growth and fibronectin production. These effects may, in part, account for the reported beneficial effects of heparin on the course of renal disease in experimental animals.     

Distribution of epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse

Clara cells are primary targets for metabolically activated pulmonary toxicants because they contain an abundance of the cytochrome P450 monooxygenases required for generation of toxic metabolites. The factors that regulate bronchiolar regeneration after Clara cell injury are not known. Previous studies of naphthalene-induced bronchiolar injury and repair in the mouse have shown that epithelial cell proliferation is maximal 1 to 2 days after injury and complete 4 days after injury. Proliferation is followed by epithelial re-differentiation (4 to 14 days). In this study, mice were treated with the environmental pollutant naphthalene to induce massive Clara cell injury. The distribution and abundance of three growth-regulatory peptides (epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), and transforming growth factor (TGF)-alpha) was determined immunochemically during repair of this acute bronchiolar injury. EGFR and its ligands were detected at low levels in cells throughout the lung including peribronchiolar interstitial cells, blood vessels, and conducting airway epithelium. Immediately after naphthalene injury (1 to 2 days), EGFR, EGF, and TGF-alpha are expressed in increased abundance in squamous epithelial cells of the injury target zone, distal bronchioles. These immunopositive squamous cells are detected in clumps in the distal bronchioles at the time when cell proliferation is maximal. EGFR protein expression is decreased slightly 4 to 7 days after injury and continues to decrease below control levels of abundance 14 to 21 days after injury. This down-regulation of EGFR is not reflected in a corresponding decrease in EGF and TGF-alpha protein expression, indicating that control of cell proliferation is regulated at the receptor level. Co-localization of EGFR and bromodeoxyuridine-positive proliferating cells in the same bronchiole indicates that EGFR is up-regulated within the proliferative microenvironment as well as in specific proliferating cells within the injury target zone. The coincident localization within terminal bronchioles of EGFR, EGF, and TGF-alpha to groups of squamous epithelial cells 2 days after naphthalene injury suggests that these peptides are important in up-regulating cell proliferation after Clara cell injury in the mouse.     

Crude liver membrane fractions and extracellular matrix components as substrata regulate differentially the preservation and inducibility of cytochrome P-450 isoenzymes in cultured rat hepatocytes

The influence of cell-substrata interactions on the preservation of basal or in-vivo-induced microsomal cytochrome P-450 isoenzyme contents in cultured rat hepatocytes and on the adaptive responses after exposure to phenobarbital or 3-methylcholanthrene in vitro, was investigated. Hepatocytes from untreated or phenobarbital-treated rats were cultured in serum-free, aprotinin-supplemented culture medium in 96-well microtiter plates coated with collagen type I (COL), laminin, fibronectin or crude liver membrane fractions/collagen type I (CMF/COL). Basal cell functions were characterized by measuring the total protein content and lactate dehydrogenase release. The relative contributions of CYP1A1/2, CYP2B1/2, CYP2C6, CYP2C11, CYP3A and CYP4A isoenzymes were determined with ELISA using monoclonal antibodies raised against purified cytochromes P-450 from rat liver microsomes. The characterization of the CMF revealed that contaminations with mitochondria, nuclei and lysosomes are relatively low. Among these, membranes derived from the endoplasmic reticulum appeared to be the major organelle contaminant of the CMF. The matrix components laminin, fibronectin and collagen type IV were found in appreciable amounts. Hepatocytes from untreated rats, cultured for up to nine days on CMF/COL-coated plates, retained their relative cytochrome P-450 contents at 1.5-3-fold higher levels when compared to cells cultured on COL, fibronectin or laminin. Similarly, hepatocytes from phenobarbital-treated rats preserved the contents of barbiturate-inducible CYP2B1/2 and CYP3A proteins best when cultured on CMF/COL. After exposure of hepatocytes cultured on CMF/COL to phenobarbital from days 3-6, CYP3A proteins were enhanced more than twofold and CYP2B1/2, depending on the exposure level, increased 1.3-6-fold. After exposure to 3-methylcholanthrene, a threefold increase of CYP1A proteins was found in CMF/COL and laminin cultures. These results indicate that CMF/COL, as a substratum in rat hepatocyte cultures, regulates gene expression of cytochromes P-450 isoenzymes for up to 9 days and provides a matrix which enables the cells to respond qualitatively similar to the response observed in different zones of the liver. This activity cannot be replaced by single-matrix components.     

Mechanism of initial attachment of corneal epithelial cells to polymeric surfaces

The initial attachment of cultured bovine corneal epithelial cells and stromal fibroblasts to two oxygen-containing synthetic polymers was studied. Cultured epithelial cells and stromal fibroblasts were seeded onto two oxygen-containing surfaces: 'tissue culture' polystyrene (TCPS) and a polymer film deposited by RF plasma deposition using a methylmethacrylate monomer (MMA/FEP). To establish the mechanism of cell attachment, the effect of the selective removal of the vitronectin and fibronectin from the serum used in the culture medium was tested. The attachment of cultured epithelial cells during the first 90 min of culture was reduced by 40% (TCPS)-80% (MMA/FEP) as a result of removing vitronectin from the medium. Attachment of these cells to TCPS was reduced by 85-95% when the serum was depleted of both fibronectin and vitronectin. However, depletion of fibronectin reduced cell attachment to TCPS by 20%, whilst on MMA/FEP cell attachment was equivalent, or higher, than that for intact serum. The attachment of cultured corneal stromal fibroblasts was similarly dependent on vitronectin but less dependent on fibronectin. Therefore, for the attachment of both cultured epithelial cells and fibroblasts to oxygen-containing surfaces in the presence of serum, there is a high requirement for serum vitronectin but a lesser requirement for fibronectin. The effects of the establishment of corneal epithelial cells in culture and the site of origin of the cells, were determined. Primary isolates of epithelial cells isolated from the limbal, central or peripheral regions of the cornea were less dependent on vitronectin for initial attachment to TCPS than were these cells after several passages in culture. Furthermore, the primary isolates were dramatically less responsive to vitronectin than the cultured cells. These results indicate that the mechanism of attachment of corneal epithelial cells to TCPS varies with the culture experience of the cells. Cells that are culture neophytes can employe endogenous mechanisms for the initial attachment to TCPS, whereas cells established in culture are dependent on exogenous vitronectin in order to attach.     

Adenine nucleotides modulate epithelial wound healing in vitro

BACKGROUND: Adenine nucleotides have been demonstrated to enhance structural and functional regeneration in experimental renal injury in rats. The mechanism of adenine nucleotide action have not been elucidated. The aim of this study was to characterize the effects of adenine nucleotides on intestinal epithelial wound healing in vitro. METHODS: The effects of adenine nucleotides on cell migration, cell proliferation and cell adhesion were studied in the non-transformed small intestinal epithelial cell line IEC-6 using an in vitro wounding model, a colorimetric BrdU assay and a hexosaminidase adhesion assay. RESULTS: The adenine nucleotides ADP and ATP were found to significantly stimulate epithelial cell restitution (migration) in vitro. Stimulation of epithelial restitution averaged 42% for ADP and 57% for ATP. In addition, adenine nucleotides inhibited the proliferation of rat small intestinal epithelial cells, averaging 56% for ADP and 74% for ATP. Enhancement of intestinal epithelial restitution and inhibition of epithelial cell proliferation by adenine nucleotides were mediated through transforming growth factor (TGF)-beta-independent pathways. CONCLUSION: These findings suggest that adenine nucleotides exert functional effects on intestinal epithelial cell populations and may play a role in the morphogenesis of the gastrointestinal tract and its remodeling after injury.