Controlled release of growth factors using synthetic glycosaminoglycans in a modular macroporous scaffold for tissue regeneration.

Healthy regeneration of tissue relies on a well-orchestrated release of growth factors. Herein, we show the use of synthetic glycosaminoglycans for controlled binding and release of growth factors to induce a desired cellular response. First, we screened glycosaminoglycans with growth factors of interest to determine kon (association rate constant), koff (dissociation rate constant), and Kd (equilibrium rate constant). As proof-of-concept, we functionalized an elastin-like recombinamer (ELR) hydrogel with a synthetic glycosaminoglycan and immobilized fibroblast growth factor 2 (FGF2), demonstrating that human umbilical vein endothelial cells cultured on top of ELR hydrogel differentiated into tube-like structures. Taking this concept further, we developed a tunable macroporous ELR cryogel material, containing a synthetic glycosaminoglycan and FGF2 that showed increased blood vessel formation and reduced immune response compared to control when implanted in a subcutaneous mouse model. These results demonstrated the possibility for specific release of desired growth factors in/from a modular 3D scaffold in vitro and in vivo.

Hydrogels derived from decellularized liver tissue support the growth and differentiation of cholangiocyte organoids.

Human cholangiocyte organoids are promising for regenerative medicine applications, such as repair of damaged bile ducts. However, organoids are typically cultured in mouse tumor-derived basement membrane extracts (BME), which is poorly defined, highly variable and limits the direct clinical applications of organoids in patients. Extracellular matrix (ECM)-derived hydrogels prepared from decellularized human or porcine livers are attractive alternative culture substrates. Here, the culture and expansion of human cholangiocyte organoids in liver ECM(LECM)-derived hydrogels is described. These hydrogels support proliferation of cholangiocyte organoids and maintain the cholangiocyte-like phenotype. The use of LECM hydrogels does not significantly alter the expression of selected genes or proteins, such as the cholangiocyte marker cytokeratin-7, and no species-specific effect is found between human or porcine LECM hydrogels. Proliferation rates of organoids cultured in LECM hydrogels are lower, but the differentiation capacity of the cholangiocyte organoids towards hepatocyte-like cells is not altered by the presence of tissue-specific ECM components. Moreover, human LECM extracts support the expansion of ICO in a dynamic culture set up without the need for laborious static culture of organoids in hydrogel domes. Liver ECM hydrogels can successfully replace tumor-derived BME and can potentially unlock the full clinical potential of human cholangiocyte organoids.

Uncontrolled asthmatics have increased FceRI+ and TGF-ß-positive MCTC mast cells and collagen VI in the alveolar parenchyma.

BACKGROUND: Asthma has been associated with increased collagen deposition in both conducting airways and alveolar parenchyma. Mast cells (MCs) are key effector cells in asthma and have the ability to affect collagen synthesis. However, the link between clinical control and changes in bronchial and alveolar MC phenotypes and specific collagens in controlled and uncontrolled asthma remains unknown. OBJECTIVE: To investigate MC phenotypes in correlation with deposition of specific collagen subtypes in patients with controlled and uncontrolled asthma as well as to healthy controls. METHODS: The tissue expression of IgE+ , FcεRI+ and TGF-ß+ MCs, as well as immunoreactivity of collagen I, III and VI, was assessed using immunohistochemistry on bronchial and transbronchial biopsies from controlled asthmatics (n = 9), uncontrolled asthmatics (n = 16) and healthy controls (n = 8). RESULTS: In the alveolar parenchyma, the total number of MCs, as well as the number of FcεRI+ MCs and pro-fibrotic TGF-ß+ MCTC, was significantly increased in uncontrolled asthma compared to both controlled asthma and healthy controls. The proportion of TGF-ß+ MCTC correlated positively to an increased immunoreactivity of alveolar collagen VI but not collagen I and III. Collagen VI was increased in the alveolar parenchyma of uncontrolled asthmatics compared to controlled asthmatics. Controlled asthmatics had an increased deposition of alveolar collagen I. In bronchi, the immunoreactivity of collagen I was increased in both controlled and uncontrolled asthmatics while collagen III was increased only in controlled asthmatics. CONCLUSIONS: Patients with uncontrolled atopic asthma have an altered pro-fibrotic MCTC phenotype in the alveolar parenchyma that is associated with alveolar collagen VI. The present data thus support distal lung mast cell and matrix changes as histopathological features of asthma that may be of particular clinical relevance in patients who have remaining symptoms despite conventional inhaler therapy.

Labour induces increased concentrations of biglycan and hyaluronan in human fetal membranes.

OBJECTIVE: The proteoglycan decorin stabilizes collagen whereas biglycan and hyaluronan disrupt well-organized collagen. The aim was to compare hyaluronan and proteoglycans in human fetal membranes obtained before and after spontaneous labour at term. STUDY DESIGN: Prelabour samples of fetal membranes (N=9) were obtained from elective caesarean sections and regionally sampled from over the cervix (cervical membranes) and mid-zone samples between this area and the placental edge. Postlabour samples (N=11) were obtained from spontaneous vaginal delivery and also regionally sampled. Amnion and chorio-decidua were analysed separately. The proteoglycans decorin and biglycan were analysed using alcian blue precipitation, SDS polyacrylamide gel electrophoresis and immunostaining. Hyaluronan was analysed using a radioimmunoassay and by histochemistry. Collagen was measured by estimating hydroxyproline content. RESULTS: In prelabour membranes the biglycan concentration (microg/mg wtw) in the cervical amnion was 40% lower than in the mid-zone amnion (P<0.05). After delivery the cervical amnion showed a twofold increase in biglycan (P<0.05), a 30% decrease in collagen (P<0.05), and a 50% decrease in decorin concentration (P<0.05). In mid-zone samples after delivery the concentrations of hyaluronan showed an increase form 1.0 to 4.9 microg/mg wtw (P<0.05). Histology demonstrated a gelatinous substance, which separated amnion and chorio-decidua, in particular at the cervical site. This gelatinous substance contained hyaluronan at a concentration of 3.0 microg/mg wtw. CONCLUSION: It is well established that prelabour fetal membranes are considerably stronger than postlabour fetal membranes. Two features may explain this; a weakening of the amnion combined with a separation of amnion and chorio-decidua. The biomechanical changes are consistent with the decrease in collagen and decorin, and the increase in hyaluronan and biglycan demonstrated in this study. The separation of the membranes is caused by the formation of a gelatinous substance, rich in hyaluronan. The results indicate that the biomechanical changes are not merely secondary to the stress of labour but that an active maturation process is involved.

BALF-derived fibroblasts differ from biopsy-derived fibroblasts in systemic sclerosis.

Growth of fibroblasts from bronchoalveolar lavage fluid (BALF) in patients with systemic sclerosis (SSc) has previously been described. The purpose of the present study was to characterise fibroblasts from BALF and bronchial biopsies from SSc patients with alveolitis and from controls, to analyse fibroblast proliferation, migration, stress fibres and proteoglycan production. BALF and bronchial biopsies were collected from 10 patients with SSc and alveolitis and from 15 controls. Outgrowth of fibroblasts was observed from the BALF of four patients, particularly in those with a markedly increased percentage of eosinophils in BALF, but not in any member of the control group. Increased levels of granulocyte-macrophage colony-stimulating factor, correlating with the percentage of eosinophils in BALF, were found in patients when compared with controls. Fibroblasts from BALF showed an elongated, mobile phenotype and increased proteoglycan production compared to the corresponding biopsy fibroblasts. In conclusion, outgrowth of fibroblasts with an altered phenotype is reported from bronchoalveolar lavage fluid in systemic sclerosis patients with alveolitis and an increased percentage of eosinophils in the bronchoalveolar lavage fluid. These findings indicate a possible role for eosinophil-fibroblast interaction in pulmonary fibrosis in systemic sclerosis.

The association between changes in skin echogenicity and the fibroblast production of biglycan and versican in systemic sclerosis.

OBJECTIVE: To investigate a possible association between the longitudinal changes in skin involvement and the fibroblast production of proteoglycans in vitro, among patients with early and untreated systemic sclerosis (SSc). METHODS: In 11 patients, 6 with diffuse cutaneous systemic sclerosis (dSSc) and 5 with limited cutaneous systemic sclerosis (ISSc), and in 6 controls skin thickness and skin echogenicity of the forearm was measured by high frequency (20 MHz) ultrasound. A skin biopsy was taken from the area of the ultrasound measurements, and from cultivated fibroblasts the production of the proteoglycans versican, perlecan, biglycan and decorin were measured. To investigate longitudinal changes in skin involvement, the ultrasound examination was repeated after 1-3 years. RESULTS: Compared to controls, SSc patients had increased skin thickness at the first evaluation. Patients with dSSc had lower skin echogenicity than both patients with lSSc and the controls. Patients with greater changes in skin thickness and skin echogenicity produced more versican, whereas the production of biglycan and decorin was higher only in patients with greater changes in skin echogenicity. There was a negative correlation between fibroblast production of biglycan and disease duration. CONCLUSION: High fibroblast synthesis of the proteoglycans versican and biglycan is associated with changes in skin echogenicity and may predict more progressive skin sclerosis in SSc.

Differences in the uptake and nuclear localization of anti-proliferative heparan sulfate between human lung fibroblasts and human lung carcinoma cells.

Heparan sulfate inhibits the proliferation of normal human lung fibroblasts (HFL-1) but not of a human lung carcinoma cell-line (A549). In this study we investigated possible mechanisms and structural requirements by which antiproliferative heparan sulfates exerts its effects on binding, uptake and subcellular localisation. Both HFL-1 and A549 cells were incubated with 125I- or rhodamine-labeled L-iduronate-rich antiproliferative heparan sulfate species as well as L-iduronate-poor inactive ones. The antiproliferative heparan sulfate was bound to the cell surface on both HFL-1 and A549 cells, but to a lesser extent and with less affinity to A549 cells. Both cell types bound the antiproliferative heparan sulfate with one high- and with one low affinity site. The L-iduronate-poor heparan sulfate bound to a lesser extent and with less affinity to both cell types compared to the antiproliferative heparan sulfate. The antiproliferative heparan sulfate accumulated in the cytoplasm of HFL-1 cells after 24 h incubation, but after 72 h it was found evenly distributed in the nucleus. The time-scale for antiproliferative activity correlated with nuclear localization. In contrast, in A549 cells it was only found near the nuclear membrane. The inactive heparan sulfate was taken up in considerably smaller amounts compared to the antiproliferative heparan sulfate and could not be detected in the nucleus of either HFL-1 or A549 cells. Our data suggest that the antiproliferative activity of L-iduronate-rich heparan sulfate on normal fibroblasts may be due to direct effects on nuclear processes, such as gene transcription.

A proteomic approach to mimic fibrosis disease evolvement by an in vitro cell line.

Subepithelial fibrosis in asthma involves an increase in the thickening of the lamina reticularis and is due to increased deposition of collagen I, III and V, and fibronectin. The cause of the thickening of the reticular layer is not known in detail, however, it is proposed to be caused by bronchial myofibroblasts. The transformation of fibroblasts to myofibroblasts may be contributed by inflammatory cytokines. In this paper we have studied and compared in vivo tissue material with a human fibroblast target cell. A normal primary fetal fibroblast cell line and HFL-1 (human fibroblast lurg cells) were used as a comparison between fibroblasts from human central biopsies regarding morphology and cell proliferation. Both cell morphology and cell proliferation rate was similar between the different set of cell cultures. Furthermore, it could be concluded that fibroblasts cultures from patients with asthma were surrounded by more extracellular matrix molecules compared to the primary cell line HFL-1, which may mimic the in vivo situation during formation of fibrosis. We wanted to investigate if differential protein display by two-dimensional (2-D) gel electrophoresis and subsequent protein identification by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry could reveal proteins induced by cytokine stimulation that can be correlated to the transformation of normal human fetal lungs cells into a more myofibroblast like phenotype. After stimulation with transforming growth factor-beta (TGF-beta) several myofibroblast markers were found to be regulated. Especially cytoskeletal and cytoskeletal-associated proteins like actin isoforms and tropomyosin, proteins that are responsible for contraction as well as transportation of extra cellular matrix proteins, which are overproduced in the formation of fibrosis. These results indicate that TGF-beta, which is increased in a fibrotic process, participates in the transformation of fibroblasts to myofibroblasts.

Proteomics -- the protein expression technology to study connective tissue biology.

During the formation of peribronchial fibrosis in asthma, remodeling of connective tissue is due to an increase in deposition of extracellular matrix components like that of specific types of collagens and proteoglycans. By taking bronchial biopsies, we were able to isolate cell cultures derived from asthmatic patients and healthy volunteers, which provides a good model system to study differences regarding cell morphology and key connective tissue proteins in the remodeling process. Proteomics, utilizing two-dimensional electrophoresis and modern image analysis systems have made it possible to study protein expression and regulation of proteins in biological systems. By using this powerful tool, it is possible to quantitatively study protein regulation and to obtain increased knowledge about the mechanism behind the inflammatory process and formation of peribronchial fibrosis. We have optimized a proteomic protocol enabling detailed investigation of the protein expression pattern in human lung cells. An increased expression pattern was obtained, whereby 20 protein spots could be detected by image analysis in the <45 kDa region. Out of these, specific regulations of four spots were found by quantitative image analysis and spots of interest were identified by MALDI TOF-MS. This protocol enables us to study 1000--2000 proteins simultaneously and the possibility to correlate protein expression to the physiological status of the cell culture investigated. We have found that two proteins, actin and tropomyosin, are increased in expression due to transforming growth factor-beta stimulation. These proteins are correlated to the transformation of normal fibroblasts to myofibroblasts which are involved in the remodeling processes observed in asthma.

Human cervical ripening, an inflammatory process mediated by cytokines.

An extensive remodelling process, referred to as cervical ripening, takes place in the cervical tissue during pregnancy and labour. It is recognized as softening and dilation of the cervical canal, and starts as a slow process during pregnancy, becoming rapid close to partum. In this study we focus on cytokines as possible mediators of this final remodelling. mRNA levels for interleukin (IL)-8, IL-6 and granulocyte colony-stimulating factor (G-CSF) were upregulated in the ripe postpartum cervical tissue (n = 8) compared to the unripe state (n = 9). Likewise, released cytokine concentrations increased from non-pregnant (n = 11) to the term-pregnant group (n = 13) with a further increase at partum (n = 16). IL-8 concentrations increased 4-fold from non-pregnant to term-pregnant (P<0.01), and a further 10-fold to postpartum state (P<0.0001). Concentrations of IL-6 and G-CSF were similarly increased. Specific IL-8 immunostaining was identified in the epithelia of pregnant cervical tissue (n = 7) and was most pronounced in the epithelia and stroma of postpartum tissue (n = 4). In conclusion, IL-8, IL-6 and G-CSF increase in the human cervix during the ripening process, indicating their important role in the cervical remodelling. These data demonstrate that cervical ripening is similar to an inflammatory process.

Alteration of proteoglycan synthesis in human lung fibroblasts induced by interleukin-1beta and tumor necrosis factor-alpha.

Important constituents of extracellular matrix are collagen, fibronectin, hyaluronan, and various types of proteoglycans, such as versican, perlecan, decorin, and biglycan. Remodeling of extracellular matrix occurs continuously and is affected by various cytokines. The aim of this study was to investigate how interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), separately and in combination, alter fibroblast proliferation, as well as the production of extracellular matrix molecules produced by human fibroblasts from lung. Fibroblast proliferation was inhibited significantly by all treatments, by 12% with IL-1beta and by 16% with TNF-alpha. The combination of IL-1beta and TNF-alpha increased the inhibition further, by 27%. Hyaluronan production was increased by all treatments: 1.7-fold by IL-1beta and 1.5-fold by TNF-alpha. The combination of the two gave a further increase (2.5-fold). Similarly, the production of total proteoglycans was increased. The small proteoglycans, decorin, and biglycan, were regulated differently. Decorin production was inhibited by about 34% by all treatments, while biglycan was upregulated 1.3-fold by TNF-alpha. Versican was upregulated by IL-1beta (1.7-fold), whereas TNF-alpha was without effect. Perlecan was mostly unaffected. The changes in protein production of the various proteoglycans were due to increased transcription, as mRNA levels were also changed to the same extent. Synthesis of mRNA for collagen type I was inhibited by up to 75% with the IL-1beta/TNF-alpha combination. The separate cytokines also decreased the level of collagen type I mRNA, but to a lesser extent: 60% with IL-1beta and 40% with TNF-alpha. In summary, our study indicates that these proinflammatory cytokines affect the regulation of extracellular matrix production, which is of importance for the inflammatory process.

Heparan sulfate upregulates platelet-derived growth factor receptors on human lung fibroblasts.

Heparan sulfate is a molecule that possesses a large structural variability and which has been shown to inhibit the proliferation of fibroblasts in vitro. The aim of this study was to determine whether the anti-proliferative effects of heparan sulfate were exerted by regulation of the activity of the platelet-derived growth factor and/or of the platelet-derived growth factor receptors. Both l-iduronate-rich, anti-proliferative and the l-iduronate-poor, non-anti-proliferative heparan sulfate species, were incubated with confluent human embryonic lung fibroblasts for 24 h. The mRNA levels for PDGF-AA, PDGF-BB, and their receptors were measured. Binding studies were performed with [125I]-PDGF-BB and [125I]-EGF for 2 h at 4 degreesC in cultures preincubated with both types of heparan sulfate for 24 h. In separate experiments, cultures were incubated together with heparan sulfate and [125I]-PDGF-BB for 2 h at 4 degreesC. Increases of two- to threefold in the mRNA levels for both the alpha- and the beta-receptors of PDGF was obtained after treatment with both types of heparan sulfate, whereas the mRNA levels of both the PDGF-AA and the PDGF-BB were essentially unaffected. A sixfold increase in binding was only noted for [125I]-PDGF-BB in cultures pre-treated with the anti-proliferative heparan sulfate for 24 h, whereas no effect was noted with use of the non-anti-proliferative heparan sulfate. Incubating the [125I]-PDGF-BB and the anti-proliferative heparan sulfate together for 2 h resulted in a smaller, threefold increase in binding. This indicates that the anti-proliferative heparan sulfate both stabilizes and increases expression of the PDGF receptors. To investigate whether the increased number of PDGF receptors could affect cell activity, cells were preincubated with anti-proliferative heparan sulfate and then treated with PDGF-BB. This resulted in an increase in mitogenicity compared to cells treated only with PDGF-BB. Neither an increase in binding for [125I-EGF] nor an increase in the mitogenic response of EGF could be observed in cultures pre-treated with the anti-proliferative heparan sulfate. The results indicate that the extracellular matrix itself may regulate important biological phenomena such as cell proliferation and matrix production through affecting the expression of receptors of PDGF, which initiate both stimulatory and inhibitory signals.

Cadmium inhibits proteoglycan and procollagen production by cultured human lung fibroblasts.

Chronic inhalation of cadmium at the workplace or in cigarette smoke is associated with emphysema, a disease characterized by extensive disruption of lung connective tissue. We have previously shown that cadmium, at noncytotoxic doses, inhibits fibroblast procollagen production in vitro, with maximal inhibitory effects of 69 +/- 6% (P < 0.01) at 30 µM cadmium chloride (CdCl2). In this paper we show that at similar doses, cadmium also inhibits proteoglycan synthesis, with values reduced by between 36 +/- 4% (P < 0.01) and 42 +/- 6% (P < 0.01) for proteoglycans secreted into the culture media and associated with the cell layer, respectively. The greatest inhibition was obtained for the major matrix-associated proteoglycans, versican, decorin, and the large heparan sulfate proteoglycans, with synthesis values reduced by between 60 and 70%. Biglycan and other heparan sulfate proteoglycans were also affected, with synthesis values reduced by between 25 and 45%. In contrast, total protein synthesis was unaffected. Furthermore, effects of cadmium at the protein level were mirrored by reduction in messenger RNA levels for alpha1(I) procollagen, versican, and decorin. These data support the hypothesis that cadmium may play an important role in the pathogenesis of emphysema associated with chronic inhalation of cadmium fumes by inhibiting the production of connective tissue proteins.

Differential expressions of mRNA for proteoglycans, collagens and transforming growth factor-beta in the human cervix during pregnancy and involution.

During pregnancy and involution, an extensive remodelling of the human cervical connective tissue occurs. This cervical ripening is one of the most pronounced physiological remodelling processes known in human connective tissue. To investigate how the remodelling is accomplished, the levels of mRNA for collagen I and III, versican and three small proteoglycans, biglycan, decorin and fibromodulin, were evaluated using Northern blots at different stages of cervical ripening. In the corresponding biopsies the concentration of collagen and of small and large proteoglycans were determined. The role of transforming growth factor-beta (TGF-beta) as a mediator of the remodelling process was also investigated. The concentration of collagen decreased and 1 week before partus, 50% of the nonpregnant level was attained. No further decrease was noted after partus. The mRNA for collagen I and III did, however, not decrease in the term pregnant cervix 1 week before partus. Only 20-30% decrease during the final ripening just before partus was recorded. Neither did the mRNA levels of the small proteoglycans change significantly during the ripening, despite an almost 50% decrease in the concentration of the small proteoglycans. The message for versican was, however, 5-fold increased at partus and then gradually returned to nonpregnant levels within 4 days after delivery. These changes corresponded to similar changes in the concentration of the large proteoglycan. Thus, the remodelling of the cervical connective tissue is achieved by two different mechanisms, on one hand an increased turnover of collagen and the small proteoglycans, on the other a changed transcription followed by an increased production of versican. During the involution 2- to 3-fold increases in the messages for collagen I and III, and the small proteoglycans, biglycan and decorin, corresponded to increases in the concentration of the small proteoglycans and non-extractable collagen. The message for TGF-beta was increased 2-fold immediately after delivery compared with the term pregnant state. Thus, TGF-beta may be of importance for the reconstruction of the cervix, which starts immediately after partus.

Different organization of collagen fibrils in stress-incontinent women of fertile age.

OBJECTIVE: The objective was to test the hypothesis that stress urinary incontinence in women is correlated to changes in the paraurethral connective tissue ultrastructure and metabolism. METHODS: Transvaginal biopsies were obtained from the paraurethral connective tissue in women of fertile age with stress urinary incontinence and in matched continent controls. All the stress-incontinent women were characterized with urodynamic investigation. In the biopsies, collagen concentration, measured as hydroxyproline, and the degree of extraction by pepsin digestion were quantified. Proteoglycan composition and concentration were analyzed using Alcian blue precipitation, followed by electrophoretic separation and quantification. Using Northern blots mRNA levels for the collagens I and III, the small proteoglycans decorin and biglycan, and the large proteoglycan versican, were quantified. Collagen organization was examined with transmission electron microscopy and the diameters of collagen fibrils were analyzed with an interactive image analysis system (IBAS, Zeiss/Kontron). RESULTS: The biochemical and morphological analyses exposed a significant difference in the paraurethral connective tissue between stress urinary incontinent women before menopause and comparable controls. The collagen concentration was almost 30% higher and the diameters of the collagen fibrils were 30% larger in the incontinent group of women. Also the organization of the collagen fibrils differed, with considerably higher cross-linking. A higher level of mRNA for collagen I and III in the incontinent group indicates that the differences can be related to an altered collagen metabolism. No change of proteoglycan amount or composition was observed, resulting in a significantly lower proteoglycan/collagen ratio in the incontinent group of women. CONCLUSION: Stress urinary incontinence in fertile women is associated with a change in collagen metabolism resulting in an increased concentration of collagen and larger collagen fibrils. These alterations should result in a more rigid form of extracellular matrix, suggesting a connective tissue with impaired mechanical function.

Paraurethral connective tissue in stress-incontinent women after menopause.

OBJECTIVE: To study whether stress urinary incontinence after menopause is correlated to changes in the paraurethral connective tissue ultrastructure and metabolism. METHODS: Transvaginal biopsies were obtained from the paraurethral connective tissue in stress urinary incontinent women after menopause with and without estrogen replacement therapy, and from comparable controls. All the stress-incontinent women underwent urodynamic investigation. In the specimens, collagen concentration, measured as hydroxyproline, and the degree of extractability by pepsin digestion, were quantified. Proteoglycan composition and concentration were analyzed using Alcian Blue precipitation, followed by electro-phoretic separation and quantification. Using Northern blots, mRNA levels for the collagens I and III, the small proteoglycans decorin and biglycan, and the large proteoglycan versican, were quantified. Collagen structure was examined with transmission electron microscopy, and the diameters of collagen fibrils were analyzed with an interactive image analysis system (IBAS, Zeiss/Kontron). RESULTS: No significant difference in paraurethral connective tissue biochemistry or ultrastructure was registered between women with stress incontinence and controls. Estrogen replacement therapy resulted in a lower collagen concentration both between the controls (p = 0.02) and between the incontinent women (0.02). In the women with stress incontinence also the extractability by pepsin digestion was higher in the group with estrogen treatment (p = 0.004), indicating a decrease in cross-linking. The proteoglycan/collagen ratio was higher in the control group with estrogen treatment compared to untreated (p = 0.02), but no difference was found between estrogen treated and untreated incontinent women. The median collagen fibril diameter was 15% larger in the incontinent group of women without estrogen therapy compared to the control group and 5% larger when comparing the incontinent group on estrogen replacement therapy to the corresponding control group. CONCLUSION: The extracellular matrix of paraurethral connective tissue in stress urinary incontinent women after menopause reacted differently to estrogen replacement therapy compared to continent controls. In contrast to incontinent women of fertile age no major changes in collagen metabolism were found in stress urinary incontinent women after menopause.

Heparan/chondroitin/dermatan sulfate primer 2-(6-hydroxynaphthyl)-O-beta-D-xylopyranoside preferentially inhibits growth of transformed cells.

Xylose forms the direct carbohydrate-protein link in extra- or pericellular proteoglycans (PGs) that are substituted with either chondroitin sulfate (CS)/dermatan sulfate (DS) and/or heparan sulfate (HS). Cell surface PGs carrying HS are important regulators of cell growth. Xylose coupled to an aromatic compound can enter cells and initiate either CS/DS synthesis or both HS and CS/DS synthesis, depending on the nature of the aromatic adduct. Here, we show that 2-(6-hydroxynaphthyl)-O-beta-D-xylopyranoside, which can prime both types of glycan chains, inhibits growth of a set of normal and transformed cells. Transformed cells are preferentially inhibited, and at a concentration of 0.15-0.20 mM xyloside, transformed cells are totally growth arrested, whereas normal cells are only < or = 50% inhibited. No inhibition of growth is observed with the stereoisomeric 2-(6-hydroxynaphthyl)-O-beta-L-xylopyranoside, which does not prime glycosaminoglycan synthesis at all; with the nonhydroxylated 2-naphthyl-O-beta-D-xylopyranoside, which only primes CS/DS synthesis under these conditions; or with p-nitrophenyl-O-beta-D-xylopyranoside, which is known to prime only CS/DS synthesis. We conclude that growth inhibition is due to priming of HS and/or CS/DS synthesis, which may either lead to the formation of specific antiproliferative glycans or glycan fragments or to interference with endogenous PG synthesis and turnover.

Cytokine regulation of proteoglycan production in fibroblasts: separate and synergistic effects.

We have studied the effects of cytokines, separately or in combination, on the production of proteoglycans in confluent cultures of fibroblasts. The cytokines used were the transforming growth factor-beta (TGF-beta), the platelet derived growth factor-AA (PDGF-AA), the platelet derived growth factor-BB (PDGF-BB) and the epidermal growth factor (EGF). Hyaluronan production increased in cells treated with TGF-beta, PDGF-AA and PDGF-BB. Combining pairs of factors did not contribute further to hyaluronan production, whereas the triple combination of EGF, TGF-beta and PDGF-BB induced an additional 1.9-fold increase. Proteoglycan production was only increased by TGF-beta alone. As for hyaluronan, combining pairs of the cytokines had no further effect on metabolism, whereas the combination of EGF, TGF-beta and PDGF-BB induced a further 1.6-fold increase in production and secretion. Compared with the control, an extensive increase in proteoglycan production was generated by the combination of EGF, TGF-beta and PDGF-BB, 7-fold for biglycan, approximately 5-fold for versican and hyaluronan and 2.4-4-fold for heparan sulfate proteoglycan and decorin. Compared with TGF-beta alone, this combination increased, in falling order, the production of heparan sulfate proteoglycan, hyaluronan, biglycan, decorin and versican. The mRNA levels for the various proteoglycans did not completely agree with the changes in production, suggesting that changes not only in synthesis but also in rate of degradation generate these variations. The data indicate that cytokines cooperate to produce a proper and physiological response, one needed by the organism during physiological and pathophysiological remodeling.

Binding, internalization, and degradation of antiproliferative heparan sulfate by human embryonic lung fibroblasts.

Binding, internalization, and degradation of 125I-labeled, antiproliferative, or nonantiproliferative heparan sulfate by human embryonic lung fibroblasts was investigated. Both L-iduronate-rich, antiproliferative heparan sulfate species as well as L-iduronate-poor, inactive ones were bound to trypsin-releasable, cell-surface sites. Both heparan sulfate types were bound with approximately the same affinity to one high-affinity site (Kd approximately 10(-8) M) and to one low-affinity site (Kd approximately 10(-6) M), respectively. Results of Hill-plot analysis suggested that the two sites are independent. Competition experiments with unlabeled glycosaminoglycans indicated that the binding sites had a selective specificity for sulfated, L-iduronate-rich heparan sulfate. Dermatan sulfate, which is also antiproliferative, was weakly bound to the cells. The antiproliferative effects of heparan and dermatan sulfate appeared to be additive. Hence, the two glycosaminoglycans probably exert their effect through different mechanisms. At concentrations above 5 micrograms/ml (approximately 10(-7) M), heparan sulfate was taken up by human embryonic lung fibroblasts, suggesting that the low-affinity site represents an endocytosis receptor. The antiproliferative effect of L-iduronate-rich heparan sulfate species was also exerted at the same concentrations. The antiproliferative species was taken up to a greater degree than the inactive one, suggesting a requirement for internalization. However, competition experiments with dextran sulfate suggested that both the high-affinity and the low-affinity sites are involved in mediating the antiproliferative effect. Structural analysis of the inactive and active heparan sulphate preparations indicated that although sulphated L-iduronate appears essential for antiproliferative activity, it is not absolutely required for binding to the cells. Degradation of internalized heparan sulfate was analyzed by polyacrylamide gel electrophoresis using a sensitive detection technique. The inactive species was partially degraded, whereas the antiproliferative one was only marginally affected.