Extracellular matrix deposition, lysyl oxidase expression, and myofibroblastic differentiation during the initial stages of cholestatic fibrosis in the rat

Early studies showed that during hepatic fibrosis induced by bile duct ligation, fibroblasts within the portal tracts proliferate and express alpha-smooth muscle (SM) actin, suggesting that they may be involved in the deposition of extracellular matrix components in cholestatic fibrosis. Thus, we investigated the deposition of extracellular matrix components (laminin, fibronectin EIIIA, collagen I and IV, procollagen III, elastin, tenascin) as well as the expression of lysyl oxidase and of alpha-SM actin in the portal zone at 24, 48, and 72 hours and 7 days after ligation of the common bile duct. Rat liver tissues were processed for immunofluorescence, in situ hybridization, immunohistochemistry, and for electron and immunoelectron microscopy. At all times examined after bile duct ligation, laminin was observed essentially in the basal membrane of vessels and portal ductules. In sham-operated animals, the fibronectin EIIIA was present exclusively in vessels; at 24 hours postinjury, fibronectin EIIIA expression appeared in both the portal zone and along sinusoids. Two days after ligation, increased expressions of collagen I and IV, procollagen III, and elastin were observed within the portal zone, compared with sham-operated animals. The deposition of these components increased thereafter. Tenascin expression increased soon after bile duct ligation in stroma surrounding proliferating ductules, reaching a maximum at 48 hours; thereafter, expression was restricted to the periphery of proliferating ductules. By in situ hybridization, procollagen I and tissue inhibitor of metalloproteinase-1 mRNA expression was greatly increased in periductular areas at 24 hours postligation and remained elevated throughout the experiment. At 24 hours, a strong reactivity for lysyl oxidase appeared in the portal zone, and, as in controls, alpha-SM actin expression was restricted to vascular SM cells. In the stroma adjacent to proliferating ductules, alpha-SM actin appeared at 48 hours, and the number of alpha-SM actin-positive cells increased until the 7th day. Lysyl oxidase staining increased until 72 hours after bile duct ligation, when it was located in areas surrounding the myofibroblastic cells. At 7 days, lysyl oxidase expression was restricted around myofibroblastic cells present at the periphery of the reactive tissue and appeared to extend into the surrounding parenchyma. These results show that after bile duct ligation, extracellular matrix deposition, and lysyl oxidase expression occur very early in portal connective tissue surrounding proliferating ductules, and precede myofibroblastic differentiation, ie, alpha-SM actin expression. In addition, the data are compatible with the suggestion that in the bile duct ligation model, myofibroblastic differentiation represents an adaptive response to modification of the extracellular matrix environment.     

Use of the Taylor Visagraph II system to evaluate eye movements made during reading

The clinical utility of transjugular intrahepatic portasystemic shunts (TIPS) is frequently complicated by the ingrowth of tissue into the stent lumen, causing stent stenosis. These studies were undertaken to define the cellular and matrix components of the pseudointima, define the phenotype and function of the mesenchymal cells in the pseudointima and maintain them in culture, and to study the differences between stenotic and nonstenosed stents. A total of 35 stents were evaluated. TIPS pseudointima were examined histologically, by immunohistochemistry and in situ hybridization to determine the cellular and connective tissue constituents. Mesenchymal cells were grown from tissue within the TIPS and around it, and their phenotype was studied and compared with control smooth muscle cells and fibroblasts. Masson's trichrome staining of histological sections demonstrated that TIPS tissue was composed of collagen and palisades of mesenchymal cells and was lined by an endothelium. Immunostaining demonstrated strong and uniform alpha-smooth muscle staining in TIPS mesenchymal cells and peri-TIPS cells. Type I procollagen mRNA expression was demonstrated in mesenchymal cells in and around the stent by in situ hybridization. TIPS mesenchymal cells secreted less radiolabeled fibronectin, and far more type III, relative to type I, collagen compared with peri-TIPS cells. TIPS cells also expressed high levels of type III procollagen mRNA compared with peri-TIPS cells. There was no difference between stenotic stents and nonstenosed stents with respect to clinical features, time from stenting, gross morphology, histology, presence of bile fistulae, and cell phenotype. However, smooth muscle cells (SMC) from stenotic stents demonstrated both greater cell proliferation and collagen I and III secretion compared with those from nonstenosed stents. These data demonstrate that TIPS stenosis results from an accumulation of collagen and proliferation of SMC within the stent lumen.     

Expression of collagen, interstitial collagenase, and tissue inhibitor of metalloproteinases-1 in restenosis after carotid endarterectomy

Extracellular matrix is the principal component of the fibrous caps of atherosclerotic plaques and intimal hyperplastic lesions of reconstructed arteries. Interstitial collagen form an important part of the matrix, and the balance between collagen synthesis and degradation by interstitial collagenase (matrix metalloproteinase-1, MMP-1) may determine whether plaques rupture or vessels develop stenosis. We examined type I procollagen gene expression in human atherosclerotic and restenotic carotid arteries using in situ messenger RNA (mRNA) hybridization and the expression of MMP-1 and its endogenous inhibitor (tissue inhibitor of metalloproteinases-1, TIMP-1) by immunohistochemistry. Compared with normal arteries, atherosclerotic plaques bed increased expression of immunoreactive MMP-1 and TIMP-1 with modest increase of type 1 procollagen mRNA. Early restenotic lesions (< 1.5 years) contained abundant type I procollagen mRNA but little immunoreactive MMP-1 and TIMP-1. Late restenotic lesions (> 4 years) resembled atheroma and exhibited increased immunoreactive MMP-1 and TIMP-1 as well as abundant type I procollagen mRNA. Compared with atherosclerotic plaques, type I procollagen is increased and MMP-1 is decreased in early restenotic lesions. MMP-1 and TIMP-1 expressions are upregulated in lesions with a clear atheroma. These findings suggest that the balance between proteolysis and matrix synthesis may influence both the stability of atheromatous plaques and the development of restenotic lesions.     

The regulation of collagen in fetal skin wounds: mRNA localization and analysis

The deposition of collagen in fetal skin wounds has been shown in several animal models. The authors used a radiolabeled RNA antisense probe, complementary to the mRNA for the alpha-1 chain of human procollagen type I, to assess regulation of this collagen species in fetal and adult rabbit wounds. Dorsal skin wounds were placed on fetal and maternal animals at the beginning of the third trimester, and were harvested 3, 5, and 7 days later. In situ RNA/RNA hybridization was performed on suitable specimens, and morphometric analysis was carried out with a computerized LECO image analyzer. Fetal wounds exhibited an inflow of mesenchymal cells that produced collagen type I at levels higher than the surrounding tissue; this activity was highest on days 3 and 5 after wounding. Adult wounds had increased fibroblast presence by day 7, producing collagen type I at levels higher than those of adjacent unwounded tissue. Morphometric analysis of the signal produced by in situ hybridization and of the number of cells producing the signal in a given field showed that fetal wounds appear to produce collagen type I by an increase in the number of cells in the area of the wound--not by induction of the gene for procollagen type I. In contrast, adult wounds had both fibroblast migration and induction of procollagen type I mRNA synthesis. These findings imply multilevel regulation of collagen production in the adult and posttranslational regulation in the fetus.     

TGF-beta1 influences early gingival wound healing in rats: an immunohistochemical evaluation of stromal remodelling by extracellular matrix molecules and PCNA

The effect of topically applied transforming growth factor beta1 (TGF-beta1) on the rat gingival wound healing process after flap surgery was evaluated by immunohistochemistry for extracellular matrix molecules (ECM), such as tenascin, heparan sulfate proteoglycan (HSPG) and type IV collagen, and for proliferating cell nuclear antigen (PCNA) in fibroblasts. TGF-beta1 solution was applied to the surgical wound experimental sites. Two microg/microl were applied at the time of the operation, and 1 microg/microl at days 1 and 2 after surgery, with contralateral control sites receiving the vehicle alone. Periodontal tissues were histologically examined at 3 and 7 days post-surgery. Tenascin was found to be more strongly stained in the granulation tissue from experimental sites at 3 days post-surgery. At 7 days postsurgery, HSPG-positive areas in granulation tissue had become smaller and there was a prominent proliferation of PCNA-positive fibroblast-like cells and type IV collagen-positive blood vessels. These results suggest that TGF-beta1 applied to surgical wounds influences early proliferation of gingival fibroblast-like cells, the formation of blood vessels, and ECM remodelling. In conclusion, TGF-beta1 application appears to promote granulation tissue formation in periodontal wound healing.     

A chemically modified tetracycline inhibits streptozotocin-induced diabetic depression of skin collagen synthesis and steady-state type I procollagen mRNA

Wasting of connective tissues including skin, bone, and cartilage have been closely associated with elevated matrix metalloproteinase (MMP) activity and depressed collagen content in the streptozotocin (STZ)-induced diabetic rat, while tetracyclines have been reported to normalize total body weight, skin hydroxyproline and collagen content in this model, in part through inhibition of MMPs. In the present study, we report the effect of CMT-1, a chemically modified tetracycline that lacks antimicrobial properties but retains divalent cation binding and MMP inhibitory activity, on diabetic skin collagen synthesis and steady-state levels of procollagen alpha 1(I) mRNA. Male, 4-month old Sprague-Dawley rats received a single injection of 75 mg/kg STZ or citrate vehicle alone and diabetic status was confirmed by positive glucosuria. Some diabetic animals received 10 mg/day of CMT-1 by oral gavage and, 28 days after STZ treatment, body weight, blood glucose values and the in vivo rates of skin collagen production were measured using the pool-expansion technique. Steady-state levels of procollagen alpha 1(I) mRNA were analyzed 21 days after STZ treatment by hybridization of total RNA with a 32P labelled cDNA to rat type I procollagen alpha 1(I) mRNA in a dot-blot assay. STZ treatment was found to significantly depress body weight, skin collagen hydroxyproline content, the in vivo rate of collagen production, and hybridizable levels of type I procollagen alpha 1(I) mRNA. CMT-1 administered daily to STZ-treated rats inhibited the diabetic depression of these parameters but had little or no effect on non-diabetic controls or on STZ-induced hyperglycemia. Thus, in addition to the inhibition of MMP mediated extracellular collagen degradation, these results suggest CMT-1 also acts to inhibit diabetic connective tissue breakdown in STZ-induced diabetes by increasing both steady-state levels of type I procollagen mRNA and collagen synthesis through mechanism(s) that are independent of the antibacterial properties of tetracyclines.     

Rapid expression and specific localization of tenascin in gastric ulcer healing in rats

This study was done to investigate the gene expression and localization of tenascin in ulcerated gastric tissues during the healing process with Northern blot analysis and immunohistochemical technique. Gastric ulcers in rats were produced by acetic acid. Tenascin mRNA levels in the ulcerated tissue were significantly increased in a biphasic manner (12 h and day 5), preceding the increase in collagen type IV and laminin mRNA levels, and returned to control levels on day 11. In intact tissues, tenascin was mainly localized in the basement membrane above the proliferative zone, in contrast to the predominant localization of collagen type IV and laminin below the proliferative zone. On the ulcer margin from 12 h to day 5, tenascin was abundantly observed in the lamina propria around nonproliferating new epithelial cells, but collagen type IV and laminin were not seen in this lamina propria. On day 7, tenascin, expressed in the lamina propria, was replaced by collagen type IV and laminin. Thus, the rapid expression and unique localization of tenascin suggest the important role of tenascin in gastric ulcer healing.     

Vascular adventitial cell expression of collagen I messenger ribonucleic acid in anti-glomerular basement membrane antibody-induced crescentic nephritis in the rabbit. A cellular source for interstitial collagen synthesis in inflammatory renal disease

BACKGROUND: Scarring in the interstitial compartment of the renal cortex heralds a poor prognosis in many forms of renal injury, however, the mechanism through which glomerular inflammation leads to interstitial scarring is not understood. In a model of anti-GBM disease in the rabbit, development of crescentic glomerulonephritis is associated with marked interstitial fibrosis and decreased renal function. We previously demonstrated that collagen accumulation in the model was preceded by increases in collagen I and IV mRNA and that these changes were primarily extraglomerular at early time points when inflammation was predominantly intraglomerular. In order to identify the cellular origins of extraglomerular collagen synthesis in this model, in situ hybridization using an alpha 2(I) procollagen probe was performed. EXPERIMENTAL DESIGN: A 602 bp rabbit alpha 2(I) procollagen cDNA was cloned using a PCR strategy and sequenced. The nucleotide sequence of the coding region was 94% identical with the human alpha 2(I) procollagen sequence. Northern blots were performed to define conditions of specific hybridization of the anti-sense riboprobe. Tissue sections from normal rabbit kidneys and from kidneys 4, 5, 7, 10 and 14 days after injection of anti-GBM antibody were hybridized with 35S-labeled sense and anti-sense riboprobes. Cells containing alpha 2(I) mRNA were identified by autoradiography and mRNA abundance was quantitated by grain density. RESULTS: No specific hybridization was detected with the sense probe at any time. alpha 2(I) mRNA was undetectable with the anti-sense probe in normal kidney sections. In contrast, the anti-sense probe hybridized specifically at all time points after induction of anti-GBM disease. In agreement with previous filter hybridization studies, on day 4, when inflammation was predominantly intraglomerular, cells in the periarterial adventitial compartment of renal cortex hybridized strongly. At later time points, labeling was also present in the interstitial spaces, the periglomerular region, in Bowman's space and in the glomerular tuft itself. CONCLUSIONS: We conclude that perivascular adventitial cells are among the first to respond to glomerular inflammation and represent a pool of cells that subsequently contribute to interstitial and glomerular scarring.     

Nitric oxide modulates basal and endothelin-induced coronary artery vascular smooth muscle cell proliferation and collagen levels

The mechanisms governing the pathological accumulation of collagen in the extracellular matrix following angioplasty are complex, but may involve interactions between endothelium-derived paracrine agents and vascular cellular components. We tested the hypothesis that nitric oxide (NO) directly decreases collagen levels and decreases endothelin (ET-1)-stimulated increases in levels of specific collagen subtypes in coronary vascular smooth muscle cells (VSMC). Cultured VSMC were incubated for 48 h with the NO donor CAS 754 (10(-4) M), ET-1 (10(-8) M), or ET-1 plus CAS 754. In some experiments, angiotensin II (Ang II; 10(-8) M) was utilized in place of ET-1. Soluble collagen types I and III were quantitated with an ELISA method, and cell counts were performed. CAS 754 significantly inhibited cell proliferation (-17+/-2% v control), basal total protein synthesis (-65+/-7% v control), and basal collagen type I levels (-39+/-6% v control), but not collagen type III levels. ET-1 and Ang II both significantly stimulated cell proliferation (26+/-5% v control), total protein synthesis (169+/-6% v control), and collagen type I levels (200+/-11% v control). Ang II, but not ET-1, significantly increased collagen type III levels. Co-incubations of ET-1 and CAS 754 resulted in a significant decrease in cell proliferation, protein synthesis, and collagen levels (-23+/-2% v control, 90+/-5% v control, and 63+/-3% v control, respectively) compared to ET-1 alone. In contrast, co-incubation of Ang II and CAS 754 had no significant effect on cell proliferation, protein synthesis, and collagen levels seen with Ang II alone. These results demonstrate that NO inhibits basal collagen levels and cell division. Additionally, NO alters ET-1 stimulation of VSMC proliferation, protein synthesis, and production of extracellular matrix components. Thus, an imbalance in key endothelium-derived compounds could significantly impact upon extracellular matrix deposition following mechanical revascularization.     

Measurement of the distribution of m-xylene in rat tissues by head space gas chromatography

Fibroblast-like cells in the periacinar region may play an important role in periacinar fibrosis. In the present study, we isolated and cultured periacinar fibroblast-like cells (PFCs) derived from human pancreatic acini and examined the characteristics of human PFCs morphologically and immunocytochemically. Immunocytochemical study of human PFCs showed that they were positively stained with antibodies against type I collagen/procollagen, type III collagen/procollagen, fibronectin, prolyl hydroxylase beta sub-unit, type IV collagen, laminin, alpha-smooth muscle actin, vimentin, and nonmuscle myosin. Electron microscopic study showed that human PFCs contained a number of microfilaments, forming dense bodies in the cytoplasm. These results indicated that human PFCs possess characteristics of myofibroblasts. Expression of alpha-smooth muscle actin, a marker of the myofibroblast-like phenotype, was increased with time in culture and was enhanced by treatment with transforming growth factor (TGF)-beta 1. Collagen synthesis in human PFCs was stimulated by TGF-beta 1 and the proliferation of human PFCs was stimulated by platelet-derived growth factor. These findings suggest that PFCs from human pancreas seem to be involved in periacinar fibrosis.     

Proliferative activity in peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous revascularization

We evaluated the proliferative activity of human atherosclerotic lesions associated with active symptoms of ischemia, by assessing the expression of the proliferating cell nuclear antigen (PCNA). We confirmed in vitro that PCNA, an essential component of the DNA synthesis machinery, is selectively expressed in proliferating human vascular smooth muscle cells. 37 atherosclerotic lesions (18 primary and 19 restenotic) retrieved by directional atherectomy from either coronary or peripheral arteries were then studied for the expression of PCNA, using in situ hybridization or immunohistochemistry. Among plaques studied by in situ hybridization, 7 out of 11 primary and 11 out of 11 restenotic lesions contained PCNA-positive cells. The mean rate of proliferation (percent of PCNA-positive cells) was 7.2 +/- 10.8% in primary lesions and 20.6 +/- 18.2% in restenotic lesions (P < 0.05). Among specimens studied by immunohistochemistry, five out of seven primary and eight out of eight restenotic lesions contained proliferating cells. The mean rate of proliferation was again higher in the restenotic (15.2 +/- 13.6%) than primary (3.6 +/- 3.5%) lesions (P < 0.05). Proliferating cells were detected as late as 1 yr after angioplasty. We conclude that cellular proliferation is a feature of atherosclerotic lesions which are associated with symptoms of ischemia, but that it is more prominent in restenosis compared to primary lesions. These findings have implications for therapies aimed at limiting lesion growth, particularly after percutaneous revascularization.     

Survey of echinococcosis in Hokkaido and measure against it

Exposure to hyperoxia results in lung injury and a decrease in lung collagen. Retinol is known to influence collagen gene expression, and retinol deficiency has been shown to potentiate hyperoxic lung injury. To investigate the combined effects of retinol deficiency and hyperoxia on lung collagen expression, retinol-deficient rats were exposed to acute hyperoxia, and expression of the alpha-1 chains of type I procollagen [pro alpha 1 (I)] and type III procollagen [pro alpha 1 (III)] were determined using Northern hybridization analyses and immunohistochemical staining. Hyperoxia alone reduced pro alpha 1 (I) mRNA by 60 +/- 4% (p < .05) and pro alpha 1 (III) mRNA by 30 +/- 5% (p < .05), and retinol deficiency alone reduced pro alpha 1 (I) mRNA abundance by 49 +/- 8.8% (p < .05) and pro alpha 1 (III) mRNA abundance by 14 +/- 7.5% (p = not significant), respectively. Retinol deficiency plus hyperoxia did not cause any further reduction in procollagen mRNA than that seen with oxygen exposure alone. Immunohistochemical staining demonstrated decreased staining for type I collagen in retinol-deficient animals. Hyperoxic exposure resulted in decreased connective tissue staining and increased alveolar wall staining for type I collagen. Retinol deficiency and hyperoxia together resulted in a marked increase in alveolar exudates staining for type I collagen. No changes in type III collagen staining were seen. These findings demonstrate that while retinol deficiency does not potentiate hyperoxia-induced reductions in procollagen mRNA, it is associated with alterations in collagen staining in distal lung and immunohistologic evidence of collagen fragments in alveolar exudates.     

Collagen type III and type IV detection in and around human hepatocellular carcinoma

Extracellular matrix proteins participate in tumor cell growth and progression. Their role in the extratumoral liver tissue needs to be elucidated. Eight patients with hepatocellular carcinoma on noncirrhotic livers are investigated by means of light microscopical and ultrastructural immunohistochemistry for collagen type III and type IV. In the tumor collagen type III, staining is weaker, and collagen type IV is increased. It is topographically located near perisinusoidal stromal cells. In the extratumoral liver tissue, the immunostaining for the two antibodies is stronger perisinusoidally. The number of Ito cells increases significantly in the extratumoral liver tissue. A lot of transitional cells are found there. Sinusoids in the extratumoral tissue are dilated and filled with lymphoid cells and platelets. The presence of matrix proteins between tumor cells is necessary to regulate their growth and differentiation. The increase in extracellular matrix content perisinusoidally in the extratumoral tissue probably erects a protective barrier against metastasizing tumor cells. There, collagen type III and type IV accumulation is probably initiated by signals coming from tumor cells, or from inflammatory cells and platelets in sinusoids.     

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.     

Collagen-synthesizing cells in initial and advanced atherosclerotic lesions of human aorta

Accumulation of extracellular matrix, fibrosis, is regarded to be one of the major manifestations of atherosclerosis. Collagen type I is the predominant matrix component in human atherosclerotic plaques. In this work we have demonstrated procollagen type I expressing cells (PCI-cells) and studied their localization in grossly normal human aorta and atherosclerotic lesions: initial lesions, fatty streaks, fibrolipid lesions (fibrolipid plaque, fibroatheroma), fibrotic lesions (fibrous plaque). PCI-cells were revealed immunocytochemically using SPI.D8 monoclonal antibody against human procollagen type I. We failed to detect PCI-cells in the areas of grossly normal aorta and media underlying atherosclerotic lesions. Positively stained cells were shown in the areas of initial lesions, fatty streaks, fibrolipid and fibrous plaques. The largest amount of PCI-cells was revealed in fatty streaks. These cells were predominantly localized in the preluminal proteoglycan-rich intimal sublayer. Intimal cells in grossly normal regions formed a common cellular network contacting each other with their processes. The cellular network is found to be partly disintegrated in atherosclerotic lesions, which leads to the appearance of isolated cells. The share of isolated PCI-cells localized outside the intimal cellular network was higher in advanced lesions than in the areas of early atherosclerotic lesions. In initial lesions most of PCI-cells were identified as smooth muscle cells using antibodies to smooth muscle alpha-actin. In fatty streaks PCI-expressing smooth muscle cells were fewer in number. Much fewer cells double-stained with anti-alpha-actin and anti-PCI antibodies were found in fibrolipid and fibrous plaques. The proportion of these double stained cells was higher among total number of PCI-cells involved in the cellular network versus PCI-cells outside the network. The results of the study demonstrated that the most active de novo synthesis of interstitial collagen takes place in the regions of atherosclerotic lesions characterized by lipid deposition, which may lead to the further progression of atherosclerotic lesions.     

Inhibition of glomerular mesangial cell proliferation and extracellular matrix deposition by halofuginone

Mesangial cell proliferation, increased deposition of collagen, and expansion of the mesangial extracellular matrix (ECM) are key features in the development of mesangioproliferative diseases. Halofuginone, a low molecular weight anti-coccidial quinoazolinone derivative, inhibits collagen type alpha 1(I) gene expression and synthesis. We investigated the effect of halofuginone on both normal and SV40 transformed mesangial cell proliferation, collagen synthesis, and ECM deposition. Proliferation of both cell types was almost completely inhibited in the presence of 50 ng/ml halofuginone. The cells were arrested in the late G1 phase of the cell cycle and resumed their normal growth rate following removal of the compound from the culture medium. The antiproliferative effect of halofuginone was associated with inhibition of tyrosine phosphorylation of cellular proteins. Similar results were obtained whether the mesangial cells were seeded on regular tissue culture plastic or in close contact with a naturally produced ECM resembling their local environment in vivo. Halofuginone also inhibited synthesis and deposition of ECM by mesangial cells as indicated by a substantial reduction in 14C-glycine and Na2(35)SO4 incorporation into the ECM, and by the inhibition of collagen type I synthesis and gene expression. It is proposed that by inhibiting collagen type I synthesis and matrix deposition, halofuginone exerts a potent antiproliferative effect that may be applied to inhibit mesangial cell proliferation and matrix expansion in a variety of chronic progressive glomerular diseases.     

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.     

Immunotherapy of head and neck cancer

Understanding the mechanisms responsible for photodamage to the skin is most important for dermatology. 3-D cultures have been used as tools to mimic the in vivo situation for several years. We irradiated such a system containing human dermal fibroblasts cultured in collagen gels, a well-known model considered to be a dermal equivalent, which reproduces the interaction between cells and the surrounding extracellular matrix. The effects of solar irradiation (315-800 nm) on the steady-state levels of the mRNAs of extracellular matrix components (type I and III collagens) and their degrading enzymes (interstitial collagenase, MMP-1 and stromelysin 1, MMP-3) were measured. Exposure to low levels of solar radiation (0-10 J cm-2 in the UVA, i.e. suberythemal UVA doses) caused a transient decrease in type I procollagen mRNA, an increase in MMP-mRNA, and no change in type III procollagen mRNA steady-state levels. These results describe the early changes in the connective tissue of the skin following exposure to low-level solar stimulation, and may help explain the long-term changes in photodamaged skin.