Changes In Glycosaminoglycan Composition Research Articles

Multivalent, calcium-independent binding of surfactant protein A and D to sulfated glycosaminoglycans of the alveolar epithelial glycocalyx.

Lung surfactant collectins, surfactant protein A (SP-A) and D (SP-D), are oligomeric C-type lectins involved in lung immunity. Through their carbohydrate recognition domain, they recognize carbohydrates at pathogen surfaces and initiate lung innate immune response. Here, we propose that they may also be able to bind to other carbohydrates present in typical cell surfaces, such as the alveolar epithelial glycocalyx. To test this hypothesis, we analyzed and quantified the binding affinity of SP-A and SP-D to different sugars and glycosaminoglycans (GAGs) by microscale thermophoresis (MST). In addition, by changing the calcium concentration, we aimed to characterize any consequences on the binding behavior. Our results show that both oligomeric proteins bind with high affinity (in nanomolar range) to GAGs, such as hyaluronan (HA), heparan sulfate (HS) and chondroitin sulfate (CS). Binding to HS and CS was calcium-independent, as it was not affected by changing calcium concentration in the buffer. Quantification of GAGs in bronchoalveolar lavage (BAL) fluid from animals deficient in either SP-A or SP-D showed changes in GAG composition, and electron micrographs showed differences in alveolar glycocalyx ultrastructure in vivo. Taken together, SP-A and SP-D bind to model sulfated glycosaminoglycans of the alveolar epithelial glycocalyx in a multivalent and calcium-independent way. These findings provide a potential mechanism for SP-A and SP-D as an integral part of the alveolar epithelial glycocalyx binding and interconnecting free GAGs, proteoglycans, and other glycans in glycoproteins, which may influence glycocalyx composition and structure.NEW & NOTEWORTHY SP-A and SP-D function has been related to innate immunity of the lung based on their binding to sugar residues at pathogen surfaces. However, their function in the healthy alveolus was considered as limited to interaction with surfactant lipids. Here, we demonstrated that these proteins bind to glycosaminoglycans present at typical cell surfaces like the alveolar epithelial glycocalyx. We propose a model where these proteins play an important role in interconnecting alveolar epithelial glycocalyx components.

Brain inflammation induces alterations in glycosaminoglycan metabolism and subsequent changes in CS-4S and hyaluronic acid

It remains uncertain how brain glycosaminoglycans (GAGs) contribute to the progression of inflammatory disorders like multiple sclerosis (MS). We investigated here neuroinflammation-mediated changes in GAG composition and metabolism using the mouse model of experimental autoimmune encephalomyelitis (EAE) and sham-immunized mice as controls. Cerebellum, mid- and forebrain at different EAE phases were investigated using gene expression analysis (microarray and RT-qPCR) as well as HPLC quantification of CS and hyaluronic acid (HA). The cerebellum was the most affected brain region showing a downregulation of Bcan, Cspg5, and an upregulation of Dse, Gusb, Hexb, Dcn and Has2 at peak EAE. Upregulation of genes involved in GAG degradation as well as synthesis of HA and decorin persisted from onset to peak, and diminished at remission, suggesting a severity-related decrease in CS and increments in HA. Relative disaccharide quantification confirmed a 3.6 % reduction of CS-4S at peak and a normalization during remission, while HA increased in both phases by 26.1 % and 17.6 %, respectively. Early inflammatory processes led to altered GAG metabolism in early EAE stages and subsequent partially reversible changes in CS-4S and in HA. Targeting early modifications in CS could potentially mitigate progression of EAE/MS.

Glycosaminoglycan-Mediated Selective Changes in the Aggregation States, Zeta Potentials, and Intrinsic Stability of Liposomes

Though the aggregation of glycosaminoglycans (GAGs) in the presence of liposomes and divalent cations has been previously reported, the effects of different GAG species and minor changes in GAG composition on the aggregates that are formed are yet unknown. If minor changes in GAG composition produce observable changes in the liposome aggregate diameter or zeta potential, such a phenomenon may be used to detect potentially dangerous oversulfated contaminants in heparin. We studied the mechanism of the interactions between heparin and its oversulfated glycosaminoglycan contaminants with liposomes. Herein, we demonstrate that Mg(2+) acts to shield the incoming glycosaminoglycans from the negatively charged phosphate groups of the phospholipids and that changes in the aggregate diameter and zeta potential are a function of the glycosaminoglycan species and concentration as well as the liposome bilayer composition. These observations are supported by TEM studies. We have shown that the organizational states of the liposome bilayers are influenced by the presence of GAG and excess Mg(2+), resulting in a stabilizing effect that increases the T(m) value of DSPC liposomes; the magnitude of this effect is also dependent on the GAG species and concentration present. There is an inverse relationship between the percent change in aggregate diameter and the percent change in aggregate zeta potential as a function of GAG concentration in solution. Finally, we demonstrate that the diameter and zeta potential changes in POPC liposome aggregates in the presence of different oversulfated heparin contaminants at low concentrations allow for an accurate detection of oversulfated chondroitin sulfate at concentrations of as low as 1 mol %.

Relaxin Regulates Hyaluronan Synthesis and Aquaporins in the Cervix of Late Pregnant Mice

Cervical ripening is associated with loss of structural integrity and tensile strength, thus enabling the cervix to dilate at term. It is characterized by changes in glycosaminoglycan composition, increased water content, and a progressive reorganization of the collagen network. The peptide hormone relaxin via interaction with its receptor, relaxin family peptide receptor 1 (RXFP1), promotes tissue hydration and increases cervical hyaluronan (HA) concentrations, but the mechanisms that regulate these effects are not known. This study in relaxin mutant (Rln(-/-)) mice tested the hypothesis that relaxin regulates HA synthase and aquaporin (AQP) expression in the cervix. We also assessed expression of the RXFP1 protein by immunohistochemistry. Pregnant Rln(-/-) mice had lower Has2 and Aqp3 expression on d 18.5 of pregnancy and decreased cervical HA compared with wild-type Rln(+/+) mice. Chronic infusion of relaxin for 4 or 6 d in pregnant Rln(-/-) mice reversed these phenotypes and increased Has2 and Aqp3 compared with placebo controls. Relaxin-treated mice also had lower Has1 and Aqp5. Changes in gene expression were paralleled by increases in cervical HA and variations in AQP3 and AQP5 protein localization in epithelial cells of Rln(-/-) cervices. Our findings demonstrate that relaxin alters AQP expression in the cervix and initiates changes in glycosaminoglycan composition through increased HA synthesis. These effects are likely mediated through RXFP1 localized to subepithelial stromal cells and epithelial cells. We suggest these actions of relaxin collectively promote water recruitment into the extracellular matrix to loosen the dense collagen fiber network.

Immunoneutralization of TGFβ1 Improves Skeletal Muscle Regeneration: Effects on Myoblast Differentiation and Glycosaminoglycan Content

When injured by crushing, the repair of the slow-twitch soleus rat muscle, unlike the fast-twitch EDL, is associated with fibrosis. As TGFβ1, whose activity can be controlled by glycosaminoglycans (GAG), plays a major role in fibrosis, we hypothesized that levels of TGFβ1 and GAG contents could account for this differential quality of regeneration. Here we show that the regeneration of the soleus was accompanied by elevated and more sustained TGFβ1 level than in the EDL. Neutralization of TGFβ1 effects by antibodies to TGFβ1 or its receptor TGFβ-R1 improved muscle repair, especially of the soleus muscle, increased in vitro growth of myoblasts, and accelerated their differentiation. These processes were accompanied by alterations of GAG contents. These results indicate that the control of TGFβ1 activity is important to improve regeneration of injured muscle and accelerate myoblast differentiation, in part through changes in GAG composition of muscle cell environment.

Diaphragm dysfunction in chronic obstructive pulmonary disease: a role for heparan sulphate?

In the present study, phage display-derived antibodies were used to investigate the topology of glycosaminoglycan epitopes in the diaphragm of chronic obstructive pulmonary disease (COPD) and non-COPD patients. Furthermore, the potential physiological significance of changes in the occurrence of glycosaminoglycan epitopes in the diaphragm of COPD patients was studied by determining the overlap in epitope recognition of glycosaminoglycan antibodies and growth factors. Diaphragm cryosections from non-COPD (n = 5) and COPD patients (Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I/II; n = 9) were incubated with antibodies directed against heparan sulphate, chondroitin sulphate and dermatan sulphate epitopes. Antibodies were visualised immunofluorescently. In addition, interference of antibody and growth factor binding with heparan sulphate epitopes was tested. Specific glycosaminoglycan epitopes showed increased expression in the diaphragm of COPD patients, whereas other epitopes were decreased or unaffected. Interestingly, the anti-heparan sulphate antibody HS4C3, which is directed against a downregulated epitope, interfered with the binding of hepatocyte growth factor. Three patients with the most severe airway obstruction also demonstrated interference of heparan sulphate antibody A04B08 with hepatocyte growth factor binding. Results indicate changes in glycosaminoglycan composition in the diaphragm of patients with chronic obstructive pulmonary disease. This may affect cellular physiology via alterations in growth factor handling and might be related to reduced levels of contractile protein in the diaphragm of these patients.

Desmoid and fibroma tumors differently respond to TGFβ 1 stimulus and ECM macromolecule accumulation

Desmoid and fibroma tumours are characterized by cell proliferation, glycosaminoglycan and collagen fibre accumulation, high levels of transforming growth factor β 1 (TGFβ 1) and different patterns of tissue infiltration. TGFβ 1 is related to extracellular matrix (ECM) composition which, in turn, regulates cell functions and cell migration. In this study we report changes in cell proliferation, glycosaminoglycan (GAG) and collagen synthesis, TGFβ 1 mRNA expression and fibronectin levels in normal, desmoid and fibroma fibroblast cultures before and after TGFβ 1 stimulation. Our data showed cell proliferation, GAG and collagen synthesis, transforming growth factor β 1 mRNA expression and fibronectin levels were significantly higher in desmoid than in fibroma cultures. TGFβ 1 treatment had no effect on cell proliferation, but increased TGFβ 1 mRNA expression, GAG, fibronectin and collagen synthesis in desmoid and fibroma fibroblasts. Its effects were more marked in desmoid cells. Fibronectin favours cell migration, while changes in GAG composition alter cell behaviour and ECM organization. In conclusion our data suggest that the different patterns of infiltration in desmoid and fibroma tumours are due to changes in ECM components and cell–ECM interactions which can be ascribed to altered TGFβ 1 mRNA expression and TGFβ 1 activity.

Age-related changes in populations of aortic glycosaminoglycans: species with low affinity for plasma low-density lipoproteins, and not species with high affinity, are preferentially affected.

Glycosaminoglycans were extracted from the intima and media layers of normal human thoracic aortas from donors of different ages. The arterial segments were devoid of macroscopically visible lesions obtained from patients who had no clinically evident cardiovascular disease. Total glycosaminoglycan content increases during the first 40 years of life. Changes in the content of hyaluronic acid and heparan sulfate are less noticeable. The content of chondroitin sulfate (mainly the 6-isomer) increases, whereas dermatan sulfate remains constant. Plasma LDL-affinity chromatography of dermatan sulfate+chondroitin 4/6-sulfate fractions allowed the separation of LDL high- and low-affinity glycosaminoglycan species. Remarkably, only glycosaminoglycan species with low affinity for plasma LDL increase with age in the disease-free areas of human thoracic aortas studied. These results suggest that age-related changes in glycosaminoglycan composition of the arterial wall do not contribute to increased deposition of plasma LDL. However, the alternative explanation that individuals with arterial glycosaminoglycans that avidly bind LDL would develop early and severe cardiovascular disease and would thus be excluded from our analysis cannot be ruled out.

Irreversible Changes in Glycosaminoglycan Composition of Anatomically Intact Bovine Articular Cartilage Induced by Intermittent Loading

The changes in matrix composition induced by I MPa intermittent (0.2 Hz) loading of anatomically intact bovine articular cartilage in vitro are studied. The kinetics of chondrocyte response was determined in experiments where sesamoid bones of adult cows were loaded for 0, 3, 5 and 7 days. The reversibility of the induced changes were studied in sesamoid bones that were loaded for 5 days and subsequently cultured without loading for another 1, 2 and 3 weeks. Water content was not affected by loading, nor by subsequent unloaded culture. Glycosaminoglycan content was not affected by loading, nor by subsequent unloaded culture for another 2 weeks. However, after 3 weeks of culture following 5 days of loading, a significant decrease in glycosaminoglycan content was found. Glycosaminoglycan synthesis was already increased after 3 days of loading. Loading for longer periods (5 and 7 days) further increased the glycosaminoglycan synthesis rate. Glycosaminoglycan synthesis decreased during the first week of subsequent culture without loading. In the third week it dropped to a lower level than observed for the control. The amount of 3-B-3(-) epitope was increased and the length of newly synthesized glycosaminoglycans was decreased by intermittent loading. Subsequent unloaded culture did not further increase the amount of 3-B-3(-) epitope. However, after 3 weeks of unloaded culture the newly synthesized glycosaminoglycans were as long as those synthesized in the control. The results suggest that the changes in glycosaminoglycan chain length were reversible. However, the overall chondrocyte response to our loading regime seems to be detrimental to the tissue: expression of 3-B-3(-) epitope induced by loading together with the drop in glycosaminoglycan content and synthesis observed at the end of the total culture period indicate that the cartilage is irreversibly damaged.

Synovial fluid glycosaminoglycan (acid mucopolysaccharide) analysis in assessment of temporomandibular joint dysfunction. A pilot study

Temporomandibular joint (TMJ) synovial fluid was aspirated from normal control subjects and patients undergoing surgery for TMJ dysfunction. The glycosaminoglycan (GAG) composition of this fluid was analysed and compared with the clinical diagnosis and histological appearance of the condylar tissues. Changes in GAG composition were observed where a histologically hyperplastic response was seen in joint tissues, but these findings did not necessarily correlate with the initial clinical diagnosis. It is suggested that the fluid composition reflects the current metabolic activities of the tissues and may provide a useful marker of such processes.

Distribution of hyaluronic acid and sulfated glycosaminoglycans during blood‐vessel development in the chick chorioallantoic membrane

This study uses histochemical methods to determine the ultrastructural distribution of specific glycosaminoglycans (GAGs) during the development of blood vessels in the chick chorioallantoic membrane (CAM) and to correlate changes in GAG composition with the significant structural events in the development of these vessels. Tissues were stained with tannic acid, ruthenium red, and high iron diamine and digested in various GAG-degrading enzymes to identify specific GAGs. The results are consistent with a role for hyaluronic acid in the formation, alignment, or migration of the capillary plexus of the CAM and a role for sulfated GAGs (heparan sulfate, chondroitin sulfate, dermatan sulfate) in the differentiation and development of arterial and venous vessels of the chorioallantoic membrane.

Glycosaminoglycans in localized scleroderma (morphoea).

The composition of glycosaminoglycans (GAGs) was analyzed in skin samples of eight patients suffering from localized scleroderma, i.e., three having generalized morphoea and five localized morphoea plaques. From each patients, biopsies were obtained from sclerotic and perilesional areas, and from clinically uninvolved skin of the same region. In the perilesional areas hyaluronic acid concentration was increased (p less than 0.05), while it was decreased (p less than 0.01) in sclerotic areas. Dermatan sulfate concentration was increased in the perilesional (p less than 0.05) as well as in the sclerotic (p less than 0.01) areas. Chondroitin 4/6 sulfate was increased in the sclerotic areas (p less than 0.05). Heparan sulfate showed no changes. No major differences were found in the total concentrations of uronic acid and hexosamine. This study demonstrates that changes in GAG composition in localized scleroderma follow previously described sequences of events of inflammation and fibrosis of connective tissue.

Incorporation of 35S-sulfate and 3H-glucosamine into heparan and chondroitin sulfates during the cell cycle of B16-F10 cells.

Changes in glycosaminoglycan composition occurring during the cell cycle were determined in B16-F10 cells sorted flow cytometrically with respect to DNA content. Incorporation of 35S-sulfate into heparan sulfate and chondroitin sulfate of unsorted and G1,S, and G2 +M sorted cells was determined following chondroitinase ABC or nitrous acid treatment; the incorporation into surface material was measured as the difference between the radioactivity of control and trypsin-treated cells. Incorporation of 35S-sulfate and 3H-glucosamine into cetyl pyridinium chloride (CPC)-precipitable material was characterized before and after chondroitinase or nitrous acid treatment by Sephadex G50 chromatography. Long-term (48 h) and short-term (1 h) labeling studies demonstrate that (a) the amount of total cellular chondroitin sulfate is greater than that of heparan sulfate, with larger amounts of unsulfated heparan than chondroitin being present; (b) the rate of turnover of heparan sulfate is greater than that of chondroitin sulfate; (c) greatest short-term incorporation of 3H-glucosamine into CPC-precipitable material occurs during S phase; and (d) the rate of turnover of both heparan sulfate and chondroitin sulfate is decreased in S phase relative to G1 and G2 + M.

Changes in glycosaminoglycan composition of normal human placentas with maturation

Crude glycosaminoglycan preparations were isolated from acetone-powder of first-, second-, and third-trimester human placentas. Glycosaminoglycan composition was examined by electrophoresis and densitometric scanning before and after treatment with testicular hyaluronidase and chondroitinase ABC. Hexosamines were determined in electrophoretic fractions in order to identify the individual glycosaminoglycans. The results showed a decrease in glycosaminoglycans and an increase in protein contents in the preparations as the placenta matured. The main glycosaminoglycans in all placentas were dermatan sulfate, chondroitin sulfates, hyaluronic acid, and heparan sulfate. With aging of the placenta, the proportion of hyaluronic acid remained unchanged and that of chondroitin sulfate decreased, whereas the proportions of dermatan and heparan sulfates increased. Age-dependent changes in the glycosaminoglycan composition of the placenta appear to follow a pattern similar to that reported for other mammalian tissues.

Changes in glycosaminoglycan composition associated with maturation of regenerating rabbit tendon

Rabbit Achilles tendons were excised and regeneration allowed to proceed for various periods of time ranging from 4 days to 4 months. Regeneration tissue was then collected and analyzed for collagen solubility, hexosamine/hydroxyproline ratios, glycosaminoglycan composition and solubility profile of the CPC complexes of the glycosaminoglycans. The most extensive changes in the glycosaminoglycan composition of the regeneration tissue took place during the first 3 weeks of regeneration, and after 4 months the composition of regeneration tissue approximated that of mature tendon. Chromatographie fractions corresponding to hyaluronate, chondroitin-4-sulfate, chondroitin-6-sulfate and dermatan sulfate accounted for 56, 11, 8 and 12% of the total CPC precipitable glycosaminoglycans in 4-day regeneration tissue, while they accounted for 20, 9, 8, and 54% in 4-month regeneration tissue and for 16, 7, 9, and 60% in mature tendon. Hexosamine/hydroxyproline ratios decreased from 0.13 in 4-day tissue to 0.02 in 4-month tissue (mature tendon, 0.02).