Initial effects of partially purified bone morphogenetic protein on the expression of glycosaminoglycan, collagen, and alkaline phosphatase in nonunion cell cultures.

Bone morphogenetic protein (BMP) stimulates mesenchymal cells to differentiate, resulting in de novo endochondral ossification in vivo. The response of fibrocartilage and periosteal cells from human and canine nonunion tissues to partially purified BMP was examined in culture. Cells derived from neonatal rat muscle explants were used for comparison. Alkaline phosphatase activity and expression of alkaline phosphatase and Types I and II collagen mRNAs were compared to that of rat chondrocytes. Synthesis of Type II collagen by the muscle cells was verified by enzyme-linked immunosorbent assay (ELISA). Addition of BMP to the muscle cell and nonunion cell cultures resulted in a dose-dependent decrease in cell number. There was a decrease in matrix vesicle and plasma membrane alkaline phosphatase activity concomitant with an increase in mRNA levels for alkaline phosphatase and collagen genes. Synthesis of immunoreactive Type II collagen increased. These data indicate that neonatal rat muscle cells and nonunion cells may respond in a similar fashion to BMP. Bone morphogenetic protein stimulated hyaluronic acid synthesis at three days, but chondroitin sulfate synthesis did not increase until ten days exposure to BMP. These data, together with those summarized above, suggest that more than three days may be required for complete expression of the chondrocyte phenotype typical of endochondral ossification.     

Effect of cyclic strain and plating matrix on cell proliferation and integrin expression by ligament fibroblasts.

The role of cell surface integrins in cell migration, proliferation, and attachment to matrix molecules is well known. Integrin-matrix interactions have been implicated in mechanotransduction and load transmission from the outside to the inside of the cell. In this study, the effect of cyclic strain on the cell proliferation, attachment, and expression of integrin subunits beta1, beta3, and alpha5 was determined in anterior cruciate ligament (ACL) and medial collateral ligament (MCL) fibroblasts grown on polystyrene, Type I collagen, laminin, elastin, and fibronectin. ACL fibroblast proliferation was not affected by growth substrate whereas MCL cells reached confluence more rapidly on fibronectin compared with collagen or polystyrene. Exposure to 5% cyclic strain resulted in a significant decrease in ACL and MCL fibroblast proliferation on fibronectin and Type I collagen. MCL cells showed a greater strain-dependent inhibition of cells grown on a fibronectin substrate than those grown on collagen. This matrix-dependent effect of strain on cell proliferation was not seen with ACL cells. Attachment of ACL and MCL fibroblasts was stronger to fibronectin compared with Type I collagen, laminin, and polystyrene. In the absence of applied load, the expression of beta1, beta3, and alpha5 subunits was not substrate dependent and the expression of beta1 and alpha5 integrin subunits was higher in MCL cells than ACL cells on all substrates. In contrast, the expression of beta3 integrin subunit was higher in ACL cells than MCL cells. In response to 5% strain, beta1, and alpha5 expression increased in all fibroblasts with MCL cells having a higher magnitude of expression. beta3 expression showed a 90% increase in response to load when grown on laminin for both MCL and ACL fibroblasts and demonstrated no change in expression on Type I collagen or fibronectin. The duration of applied strain from 2 versus 22 h had no effect on cell proliferation or integrin expression.     

Proteoglycans: the "Teflon" of the airways?

Proteoglycans are a family of structurally distinct, polyanionic complex carbohydrates composed of repeating disaccharide units. Proteoglycans include heparin, heparan sulphate, chondroitin 4- sulphate, chondroitin 6-sulphate, dermatan sulphate, and hyaluronic acid. Heparin is found in the granules of a subset of mast cells where it is bound to various mediators including histamine. Heparan sulphate has a much wider distribution in the body, being associated with stromal matrices, basement membrane and many cell surfaces, particularly the surface of endothelial cells. Heparin is an anticoagulant, but it is now very apparent that it possesses many other biological activities that have relevance to our understanding of lung diseases, particularly inflammatory diseases of the airway. Recent evidence suggests in the airway when administered by inhalation that could be exploited therapeutically.


     

The effect of glycosaminoglycans on the crystallisation of calcium oxalate

The effect of glycosaminoglycans on urinary stone formation was evaluated using a mixed suspension, mixed product removal (MSMPR) crystallisation system together with scanning electron microscopy (SEM) to examine the resulting crystals. Chondroitin sulphate was found to decrease the nucleation rate and to promote both the growth rate and suspension density. Results obtained with hyaluronic acid, although inconclusive, are similar to those given by chondroitin sulphate. Heparin sodium salt had a powerful inhibitory effect on both the nucleation rate and the suspension density, the effect increasing in proportion to the heparin concentration. SEM examination showed that the octahedral habit of calcium oxalate dihydrate was modified by the addition of heparin sodium salt and confirmed that the average crystal size in the presence of chondroitin sulphate and hyaluronic acid was significantly greater than the control or that found in the presence of heparin sodium salt.     

Human colon cancer and fibroblast cell lines cultured in and on collagen gels

Collagen is the major constituent of the in vivo extracellular matrix environment and the ability of collagen substrates to support growth of cultured cells in vitro is well recognized. The aim of the present study was to examine in vitro proliferation and matrix-binding of cells obtained from a human colon fibroblast and four colon cancer cell lines cultured in a collagen matrix environment. In contrast to colon fibroblasts, colon cancer cell lines proliferated in this culture system and their proliferative capacities were dependent upon the collagen concentration and whether tumour cells were seeded on or in the collagen. Both laminin and fibronectin stimulated growth of one of the four colon cancer cell lines without an apparent increase in cell-matrix binding. The use of collagen matrices to culture tumour cells in vitro might facilitate identification of factors which regulate growth of an individual's colorectal cancer.     

The transfer of ocular cells using collagen

The present study was designed to evaluate the use of collagen gel loaded with human retinal pigment epithelium (ARPE19) in cellular transfer and to assess its viability within the gel. Collagen solution was prepared by dissolving calfskin in hydrochloric acid to make a final concentration of 2.0 mg/ml and this was mixed with 10,000 ARPE19 cells/ml. The cell viability in gel was determined using MTT assay. van Gieson stain and proliferating cell nuclear antigen (PCNA) were used to identify the location of collagen and to localize the site of cell proliferation, respectively. The ARPE19 cells in gel appeared to be healthy with a rounded morphology. The optimal collagen concentration was 1.9 mg/ml. When this concentration was used to hold cells for over 12 days, it could be seen that the growth rate was the same between day 2 and day 8 in gel and on plastic. When the cell-loaded gels were transferred onto standard tissue culture plastics, progressive cell migrations over time resembling cell migrations in organotypic explant cultures were observed. Upon intravitreal injection of cell-containing collagen suspension into a rabbit's eye, the gel became suspended within the vitreous a few hours after injection (day 0). However, it became obvious that the gel dispersed and spread around the vitreous even after just 24 h. These cells inside the vitreous were PCNA positive, indicating that the human ARPE19 cells have the capacity to proliferate even after 11 days. The present study demonstrated the potential use of collagen gel as a tool in the transfer of cellular matrix onto other substrates. The results show that the cell seeding number must be critically balanced with the concentration of gel for it to be used as transplant material.     

The proteoglycan synthesis repertoire of rabbit chondrocytes maintained in type II collagen gels

Repair of experimental articular cartilage lesions employing cultured rabbit articular chondrocytes requires a detailed knowledge of the phenotypic stability of these cells. A suitable matrix vehicle for use in chondrocyte transplantation is a much sought-after component of any transplantation paradigm. We studied the proteoglycan synthesis repertoire of young immature rabbit articular chondrocytes maintained in chick type II collagen gels or collagen gels supplemented with recombinant human transforming growth factor-beta 1 (rhTGF beta 1). Maintenance of chondrocytes in type II collagen gels increased the percentage 35SO4-labeled proteoglycans reaching equilibrium in the A1D1 or D1 fraction of CsCl density gradient when compared to chondrocytes maintained in polystyrene microwell cultures. Although rhTGF beta 1 supplementation increased the percentage of A1D1/D1 proteoglycan by chondrocytes grown on polystyrene, rhTGF beta 1 did not augment this percentage increase in A1D1/D1 when added to collagen II gels. Rabbit chondrocytes synthesized two core proteins derived from the high-density aggregatable proteoglycans. LI and LII have apparent molecular sizes of 480 kDa and 390 kDa, respectively. Both core protein forms were found in the medium fraction, but the predominant core protein form associated with the cell fraction was LI. Maintenance of chondrocytes in collagen II gels increased synthesis of both core proteins. In addition to the large core proteins, three other core proteins with properties on SDS PAGE characteristic of the small dermatan sulfate proteoglycans, biglycan and decorin, were identified. Synthesis of these core proteins was stimulated by maintenance in collagen gels. Furthermore, they were preferentially retained in the gel matrix. Chondrocytes maintained on glass or in type II collagen gels stained with monoclonal antibodies specific for chondroitin-6-sulfate, chondroitin-4-sulfate and keratan sulfate. However, while chondrocytes grown on glass slides failed to stain with monoclonal antibody 3B3 in the absence of chondroitinase ABC digestion, chondrocytes grown in collagen II gels stained intensely in the absence of enzyme pretreatment. These results were confirmed by Western blots.     

Cyclosporine suppresses cell growth and collagen production in hepatic stellate cells

BACKGROUND: In HCV-related graft hepatitis, immunosuppression has been implicated in rapid progression to cirrhosis, a serious clinical issue. We investigated the effects of cyclosporine or tacrolimus on cell growth and collagen production by hepatic stellate cells (HSC), which play a role in hepatic fibrosis. MATERIALS AND METHODS: Cultured rat HSCs and human HSC-derived TWNT-4 cells were evaluated for proliferation, type I collagen, phosphorylation states of mitogen-activated protein kinases extracellular signal-regulated kinase 1/2; [MAPKs Erk1/2], c-Jun N-terminal kinase (JNK, p38), as well as the expression of collagen, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) genes. RESULTS: Cyclosporine suppressed cell growth and collagen production in a concentration-dependent manner. At clinically relevant concentrations of 0.125 micromol (150 ng/mL) cyclosporine significantly reduced collagen production per cell by more than 50%. Similarly, tacrolimus also reduced both collagen concentration and cell number; however, tacrolimus at a clinically relevant concentration of 12.5 nmol (10 ng/mL) did not significantly reduce collagen production. Treatment with cyclosporine reduced type I collagen and TIMP-1 expression and enhanced MMP-1 expression. Cyclosporine also inhibited phosphorylation strongly for JNK and p38, and weakly inhibited for Erk1/2. CONCLUSION: These findings demonstrated that cyclosporine suppresses cell growth and collagen production, suggesting that it may have an antifibrogenic effect.     

Synthesis of extracellular matrix components by somatic testicular cells from immature and pubertal rats

Total testicular cells derived from immature and pubertal rats were cultured under long-term conditions. Somatic adherent cells proliferated in culture and produced collagen and proteoglycans. Collagen synthesis accounted for 25% and 5% of total protein synthesized by adherent cells derived from immature, and pubertal rats, respectively. Proteoglycan synthesis was higher in cells from immature than from pubertal rats. The proportion of different types of glycosaminoglycan chains (particularly hyaluronic acid and chondroitin sulphate) also varied according to the age of the donor. The results suggest that the synthesis of extracellular matrix components by somatic testicular cells is an age-related process which probably plays an active role in spermatogenesis.     

Laminin and type IV collagen in the human testis

Specimens of normal human testis and biopsies from testes with Sertoli-cell-only syndrome in which the seminiferous tubules had a remarkably thickened lamina propria, were investigated immunohistochemically using specific antibodies against human laminin and human type IV collagen. In the normal testis, both laminin and type IV collagen were localized to the epithelial basement membranes and the peritubular cell layers. In addition, laminin was found in the Sertoli cells. In the pathological testis, structures representing invaginations of the tubular basement membrane were positive for both laminin and type IV collagen. The presence of laminin and type IV collagen in the myoid cell layers, and laminin in the Sertoli cells from both normal and pathological testis and its indication for the secretion of these substances by the myoid and Sertoli cells is discussed.     

Phenotypic modifications of human mesangial cells by extracellular matrix: the importance of matrix in the contractile response to reactive oxygen species

The progression of chronic renal diseases is characterized by the accumulation of extracellular matrix proteins in the glomerulus. The present experiments were designed to analyze the effect of hydrogen peroxide on the contractile and proliferative phenotypes of human mesangial cells grown on different culture substrates: plastic, collagen type I, and collagen type IV. Contraction was analyzed by measuring planar cell surface area and myosin light chain phosphorylation, whereas proliferation was studied by [(3)H]thymidine incorporation. No changes were detected in the proliferation rate of human mesangial cells grown on different culture substrates, neither under basal conditions nor in the presence of fetal calf serum or H(2)O(2). Cells grown on plastic or collagen did not contract in the presence of H(2)O(2), but cells grown on collagen I elicited a significant contraction with H(2)O(2). Platelet-activating factor induced contraction of human mesangial cells on the three culture substrates. The different contractile responses observed were not due to different degradation rates of H(2)O(2). The present experiments support the importance of extracellular matrix in the response to exogenous stimuli and point to the possibility that patients with changes in the mesangial matrix as a result of chronic renal diseases may have an increased susceptibility to the pathological actions of reactive oxygen species.     

The metabolic heterogeneity of glycosaminoglycans of the different zones of the epiphyseal growth plate and the effect of ethane-1-hydroxy-1, 1-diphosphonate (EHDP) upon glycosaminoglycan synthesis in vivo

The synthesis of total and unextractable glycosaminoglycans of rabbit epiphyseal growth plate was studied by determination of the incorporation in vivo of³⁵S-sulphate into various glycosaminoglycans separated by microfractionation procedures using CPC and ECTEOLA columns. In the narrow hypertrophic zone, hyaluronic acid and chondroitin sulphate of low molecular weight were found to be unextractable with 3M guanidinium chloride for 24h, thus being firmly bound within the tissue. While theunextractable chondroitin sulphate showed a sharply reduced synthesis rate in the hypertrophic zone compared with that in the columnar and resting zones, a rapid increase in the synthesis ofextractable chondroitin sulphate toward the calcification front is indicated. At microfraction, solubility profiles indicated that the unextractable chondroitin sulphate was of predominantly lower molecular weight and/or charge density compared to that of the whole tissue, most evident in the hypertrophic zone. The distribution pattern of incorporated³⁵S showed a close correspondence to the distribution of the various chondroitin sulphate fractions both for extracted and unextracted tissue. In the hypertrophic zone, higher radioactivity was found for chondroitin sulphate of predominantly low molecular weight and/or charge density in extracted compared to unextracted tissue. Low doses of EHDP caused no change of distribution of the various glycosaminoglycans but generally inhibited their synthesis.     

The metabolic heterogeneity of glycosaminoglycans of the different zones of the epiphyseal growth plate and the effect of ethane-1-hydroxy-1, 1-diphosphonate (EHDP) upon glycosaminoglycan synthesis in vivo

The synthesis of total and unextractable glycosaminoglycans of rabbit epiphyseal growth plate was studied by determination of the incorporation in vivo of³⁵S-sulphate into various glycosaminoglycans separated by microfractionation procedures using CPC and ECTEOLA columns. In the narrow hypertrophic zone, hyaluronic acid and chondroitin sulphate of low molecular weight were found to be unextractable with 3M guanidinium chloride for 24h, thus being firmly bound within the tissue. While theunextractable chondroitin sulphate showed a sharply reduced synthesis rate in the hypertrophic zone compared with that in the columnar and resting zones, a rapid increase in the synthesis ofextractable chondroitin sulphate toward the calcification front is indicated. At microfraction, solubility profiles indicated that the unextractable chondroitin sulphate was of predominantly lower molecular weight and/or charge density compared to that of the whole tissue, most evident in the hypertrophic zone. The distribution pattern of incorporated³⁵S showed a close correspondence to the distribution of the various chondroitin sulphate fractions both for extracted and unextracted tissue. In the hypertrophic zone, higher radioactivity was found for chondroitin sulphate of predominantly low molecular weight and/or charge density in extracted compared to unextracted tissue. Low doses of EHDP caused no change of distribution of the various glycosaminoglycans but generally inhibited their synthesis.     

Effect of a collagen substrate on the growth and development of normal and tumorigenic rat urothelial cells

The growth and morphology of 4 tumorigenic rat urothelial cell lines grown on collagen-coated nylon discs was characterized and compared to normal cells. In contrast to cells cultured on a plastic substrate with or without a thin "nonporous" collagen coating, tumor cells grown on porous collagen-coated nylon discs: 1) grew to greater protein densities; 2) formed tissue structures characteristic for the type of tumor they developed upon back-transplantation; and 3) could be grown and cultured indefinitely without subculturing. Thus, similarly to normal urothelial stratification and differentiation in vitro, tumorigenic cells apparently require a "porous" collagen substrate to allow differentiation analogous to that observed in vivo.     

Neutrophil-derived serine proteinases enhance membrane type-1 matrix metalloproteinase-dependent tumor cell invasion

BACKGROUND: Matrix metalloproteinase-2 degrades a variety of basement membrane components and is essential for tumor invasion. We have previously reported that membrane type-1 matrix metalloproteinase (MT1-MMP) cooperates with neutrophil-derived serine proteinases (NDPs; elastase, cathepsin G, protease-3) to activate matrix metalloproteinase-2. We therefore hypothesized that NDPs enhance tumor-cell invasion. METHODS: Clones of human HT1080 fibrosarcoma cells transfected with MT1-MMP sense (HT-SE) or antisense CDNA (HT-AS) were used. These cells express either high (HT-SE) or extremely low levels (HT-AS) of MT1-MMP relative to nontransfected HT1080 cells (HT-WT). The cells were incubated in the presence or absence of purified NDP, with or without alpha 1-antitrypsin or the MMP inhibitor batimastat. Cell invasion was measured with the use of Boyden chambers with polycarbonate membranes coated with a reconstituted extracellular matrix. RESULTS: Under control conditions HT-WT and HT-SE cells were 4-fold more invasive than HT-AS cells. The addition of NDP increased HT-WT and HT-SE cell invasion 60% to 100% but had no effect on HT-AS cells. alpha 1-antitrypsin or batimastat did not decrease the baseline invasiveness of HT-WT and HT-SE cells; however, they abrogated the stimulatory effect of NDP. CONCLUSIONS: HT1080 cell invasion depends on MT1-MMP expression. MT1-MMP overexpression does not increase invasiveness by itself. NDPs increase invasion by MT1-MMP expressing cells by activating matrix metalloproteinase-2.     

Type IV collagen,type IV collagenase activity and ability of cell proliferation in human thyroid tumours

BACKGROUND: The processes of malignant tumour invasion and metastasis are known to include the destruction of cell stroma and vascular basement membrane. It has been suggested that type IV collagenase degrades type IV collagen, a main component of the basement membrane. METHODS: In our study, type IV collagenase activity in human thyroid tumours was measured by the Liotta method. The degree of destruction of diseased regions of thyroid tumours was immunohistochemically determined by anti-type IV collagen antibody staining. Cell proliferation in the tumours was estimated using anti-proliferating cell nuclear antigen (PCNA) and epidermal growth factor receptor (EGFR). RESULTS: T4 thyroid carcinomas with higher type IV collagenase activity and very weak type IV discontinuous immunostaining for type IV collagen of follicular basement membranes, exhibited many PCNA or EGFR positive cells. In benign tumours, normofollicular- or macrofollicular-type tumours with low type IV collagenase activity showed few PCNA and EGFR positive cells and intact type IV collagen of basement membranes, as seen in normal thyroids. Conversely, an atypical adenoma with higher type IV collagenase activity showed many PCNA and EGFR positive cells and weak type IV discontinuous immunostaining for type IV collagen, as in thyroid carcinomas. CONCLUSION: These findings suggest that staining for type IV collagen and type IV collagenase activity reflect the ability of cell proliferation, and help predict the aggressiveness of invasion and metastasis in human thyroid tumours.     

Type IV procollagen mRNA regulation: evidence for extracellular matrix/cytoskeleton/nuclear matrix interactions in human urothelium.

The absence of basement membrane components correlates with tumor stage and progression in human bladder cancers. We have previously shown that invasive tumors possess the ability to degrade basement membrane. However, the presence of basement membrane may be affected not only by its degradation, but by its synthesis and deposition as well. Our results in the present study suggest that while the invasive human transitional carcinoma cell line EJ has an increased amount of type IV procollagen mRNA when compared to the non-invasive RT4 cell line, type IV collagen staining is absent in the invasive EJ cells and intensely present in the non-invasive RT4 cells. Moreover, when EJ cells were grown on an artificial basement membrane (Matrigel), type IV procollagen mRNA expression was down-regulated to the levels seen with the non-invasive RT4 cells. We also discovered that the invasive cells, when grown on Matrigel, appeared morphologically different from the same cells grown on plastic tissue cultures. We conclude that a deficient basement membrane in invasive cancer cells may be due not only to active proteolytic activity but also to an abnormal production and deposition of extracellular matrix components. In addition, we also demonstrated that basement membrane components may have a significant effect on epithelial cell morphology and gene regulation, and that any alterations of the extracellular matrix-cytoskeleton-nuclear matrix interactions can lead to altered gene regulations and cell function.     

Oxygen reduces accumulation of type IV collagen in endothelial cell subcellular matrix via oxidative stress

Anchorage-dependent cells in culture attach initially to proteins adsorbed to the culture substrate from the medium, and produce and deposit a subcellular matrix during the course of the cultivation. The aim of this study was to determine whether the concentration of O(2) in the culture atmosphere affects the accumulation of type IV collagen and laminin under human endothelial-cell monolayers. Enzyme-linked immunoassays on decellularized polystyrene substrates showed less type IV collagen, but not less laminin, under cells incubated in the standard atmosphere (5% CO(2) in air, i.e., approximately 20% O(2)) compared to an atmosphere of 5% O(2) and 5% CO(2) in N(2). Type IV collagen accumulation was inhibited via oxidative stress, because the inhibitory effect of 20% O(2) was antagonized by antioxidant ascorbic acid, and mimicked by prooxidant pyrogallol and exogenous H(2)O(2). Measurements of endogenous H(2)O(2) accumulation demonstrated that endothelial cells partially adapt to the high O(2) concentration. These results may have implications in endothelium modeling in vitro and in engineering of endothelial cell sheets and endothelialized vascular grafts.     

The alpha2 and alpha3 integrins are required for morphologic differentiation of an intestinal epithelial cell line

BACKGROUND: The molecular mechanisms controlling intestinal epithelial cell differentiation are poorly defined because of the difficulty of growing normal intestinal cells. We have taken advantage of the ability of the Caco-2 cell line to acquire a glandular phenotype in 3-dimensional (3-D) culture systems to investigate the role of alpha2 and alpha3 integrins in morphologic differentiation. METHODS: Caco-2 cells transfected with sense or antisense DNA constructs of alpha2 or alpha3 integrins were grown in 3-D Matrigel or collagen I in the presence or absence of integrin function-blocking antibodies. We used light and confocal microscopy, BrDU incorporation, TUNEL assay, a fluorometric adhesion assay, FACS analysis, and Western blot analysis to study the effect of extracellular matrix (ECM) and integrins on morphology, polarization, proliferation, apoptosis, cell adhesion, and integrin expression. RESULTS: Compared to collagen I, Caco-2 cells cultured in 3-D Matrigel display cytoskeletal and adherens junction rearrangements and decreased proliferation consistent with cellular differentiation. These changes, which are inhibited by alpha2 and alpha3 blocking monoclonal antibodies and alpha2 and alpha3 antisense DNA transfection, were associated with an increase in alpha3 integrin expression. CONCLUSIONS: We demonstrated that signaling through both constitutively expressed alpha2 integrin and Matrigel-induced alpha3 integrin expression is required to acquire a differentiated phenotype in Caco-2 cells.