Glycosaminoglycan Profile Research Articles

Structural remodeling of proteoglycans upon retinoic acid-induced differentiation of NCCIT cells

Pluripotent and multipotent cells become increasingly lineage restricted through differentiation. Alterations to the cellular proteoglycan composition and structure should accompany these changes to influence cell proliferation, delineation of tissues and acquisition of cell migration capabilities. Retinoic acid plays an important role in pre-patterning of the early embryo. Retinoic acid can be used in vitro to induce differentiation, causing pluripotent and multipotent cells to become increasingly lineage restricted. We examined retinoic acid-induced changes in the cellular proteoglycan composition of the well-characterized teratocarcinoma line NCCIT. Our analysis revealed changes in the abundance of transcripts for genes encoding core proteins, enzymes that are responsible for early and late linkage region biosynthesis, as well as enzymes for GAG chain extension and modification. Transcript levels for genes encoding core proteins used as backbones for polysaccharide synthesis revealed highly significant increases in expression of lumican and decorin, 1,500-fold and 2,800-fold, respectively. Similarly, glypican 3, glypican 5, versican and glypican 6 showed increases between 5 and 70-fold. Significant decreases in biglycan, serglycin, glypican 4, aggrecan, neurocan, CD74 and glypican 1 were observed. Disaccharide analysis of the glycans in heparin/heparan sulfate and chondroitin/dermatan sulfate revealed retinoic acid-induced changes restricted to chondroitin/dermatan sulfate glycans. Our study provides the first detailed analysis of changes in the glycosaminoglycan profile of human pluripotent cells upon treatment with the retinoic acid morphogen.

Glycosaminoglycan profiles in the uterus of adult ovariectomized rats treated with estrogen and progestagen

To evaluate the effects of conjugated equine estrogens (CE) alone or in combination with medroxyprogesterone acetate (MPA) on glycosaminoglycans (GAGs) in the cervix and horns of the rat uterus. Thirty days after ovariectomy, adult rats were randomly divided into four groups: GI, control (treated with drug vehicle); GII, CE (50 μg/kg per day); GIII, MPA (0.2mg/kg per day), and GIV, CE+MPA (doses as in GII and GIII). Drugs and vehicle were given by gavage during 28 days. Afterwards the animals were anesthetized, the cervix and uterine horns were dissected out and the middle portion fixed in 10% formaldehyde solution; other portions were fixed in acetone for histological examination and glycosaminoglycan quantification, respectively. Agarose gel electrophoresis was used for sulfated GAG analyses, and hyaluronic acid was assayed with an ELISA-like method. Statistical analysis was done by the Student's t test and the Tukey-Kramer test (P<0.05). The cervix and uterine horn structures presented signs of atrophy in the control group (GI). The other groups, mainly groups III and IV, had histological aspects of proliferation. In all groups the concentration of sulfated GAGs (especially dermatan sulfate) was higher than that of non-sulfated GAGs, both in cervix and in uterine horns. Estrogens increased sulfated GAG concentration at the cervix and the horn, whereas in uterine horns the amounts of sulfated GAGs were decreased after estrogens plus MPA treatment. The concentration of hyaluronic acid in uterine horns was higher than in cervices. The profiling and amounts of glycosaminoglycans in the two portions of the rat uterus are uneven. Dermatan sulfate occurs in higher concentrations in both cervix and uterine horns. Sulfated GAGs in rat cervix were increased by estrogens plus MPA, but were decreased by MPA alone in uterine horns.

Heparanase expression and glycosaminoglycans profile in renal cell carcinoma

A better understanding of the molecular mechanisms of renal cell carcinogenesis could contribute to a decrease in the mortality rate of this disease. The aim of this study was to evaluate the glycosaminoglycans profile and heparanase expression in renal cell carcinoma. The study included 24 patients submitted to nephrectomy with confirmed pathological diagnosis of renal cell carcinoma. The majority of the samples (87.5%) were classified in the initial stage of renal cell carcinoma (clinical stages I and II). Heparanase messenger ribonucleic acid expression was evaluated by quantitative real-time reverse transcription polymerase chain reaction, and sulfated glycosaminoglycans were identified and quantified by agarose gel electrophoresis of renal cell carcinoma samples or non-neoplastic tissues obtained from the same patients (control group). The sulfated glycosaminoglycans and hyaluronic acid were analyzed in urine samples of the patients before and after surgery. The data showed a significant statistical increase in chondroitin sulfate, and a decrease in heparan sulfate and dermatan sulfate present in neoplastic tissues compared with non-neoplastic tissues. Higher heparanase messenger ribonucleic acid expression in the neoplastic tissues was also shown, compared with the non-neoplastic tissues. The urine glycosaminoglycans profile showed no significant difference between renal cell carcinoma and control samples. Extracellular matrix changes observed in the present study clarify that heparanase is possibly involved with heparan sulfate turnover, and that heparanase and the glycosaminoglycans can modulate initial events of renal cell carcinoma development.

Mucopolisacaridosis tipo VII: ¿causa infrecuente de hidrops fetal?

We report the case of a pregnant woman who was diagnosed with hydrops fetalis at 17 weeks in two consecutive pregnancies. Once an immune origin was ruled out, the association with lysosomal storage diseases was investigated during the second pregnancy. This showed decreased activity of beta-glucuronidase in cultured cells and a glycosaminoglycan profile in amniotic fluid, indicative of Sly disease or mucopolysaccharidosis type VII which is inherited in an autosomal recessive pattern.

β-d-Xylosides Stimulate GAG Synthesis in Chondrocyte Cultures Due to Elevation of the Extracellular GAG Domains, Accompanied by the Depletion of the Intra–pericellular GAG Pools, with Alterations in the GAG Profiles

The familial disease of hereditary multiple exostoses is characterized by abnormal skeletal deformities requiring extensive surgical procedures. In hereditary multiple exostoses patients there is a shortage in the pericellular glycosaminoglycan (GAG) of heparan sulfate (HS), related to defective activity of HS glycosyltransferases, mainly in the pericellular regions of chondrocytes. This study searched for a novel approach employing xylosides with different aglycone groups priming a variety of GAG chains, in attempting to alter the GAG compositional profile. Cell cultures of patients with osteochondroma responded to p-nitrophenyl β-d-xyloside by a significant increase in total GAG synthesis, expressed mainly in the extracellular domains, limited to chondroitin sulfate). The different β-d-xylosides, in addition to increasing the synthesis of extracellular GAGs, led to a significant depletion of the intracellular GAG domains. In mouse chondrocyte cultures, β-d-xylosides with different aglycones created a unique distribution of the GAG pools. Of special interest was the finding that the naphthalene methanol β-d-xyloside showed the highest absolute levels of HS-GAGs in both extracellular and intra–pericellular moieties compared with other β-d-xylosides and with controls without xyloside. In summary, β-d-xylosides can be utilized in chondrocyte cultures to modify the distribution of GAGs between the extracellular and intracellular compartments. In addition, xylosides may alter the profile of specific GAG chains in each moiety.

Wild blueberry (V. angustifolium)-enriched diets alter aortic glycosaminoglycan profile in the spontaneously hypertensive rat

Glycosaminoglycans (GAGs) are essential polysaccharide components of extracellular matrix and cell surface with key roles on numerous vascular wall functions. Previous studies have documented a role of wild blueberries on the GAG profile of the Sprague–Dawley rat with a functional endothelium as well as in the vascular tone of the spontaneously hypertensive rat (SHR) with endothelial dysfunction. In the present study, the effect of wild blueberries on the composition and structure of aortic GAGs was examined in 20-week-old SHRs after 8 weeks on a control (C) or a wild blueberry-enriched diet (WB). Aortic tissue GAGs were isolated following pronase digestion and anion-exchange chromatography. Treatment of the isolated populations with specific GAG-degrading lyases and subsequent electrophoretic profiling revealed the presence of three GAG species, i.e., hyaluronic acid (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). A notable reduction of the total sulfated GAGs and a redistribution of the aortic GAG pattern were recorded in the WB as compared to the C group: a 25% and 10% increase in HA and HS, respectively, and an 11% decrease in GalAGs. Fine biochemical analysis of GalAGs at the level of constituent disaccharides with high-performance capillary electrophoresis revealed a notable increase of nonsulfated (18.0% vs. 10.7%) and a decrease of disulfated disaccharides (2.2% vs. 5.3%) in the WB aorta. This is the first study to report the redistribution of GAGs at the level of composition and their fine structural characteristics with implications for the endothelial dysfunction of the SHR.

Effect of the gonadal status and the gender on glycosaminoglycans profile in the lower urinary tract of dogs

Glycosaminoglycans (GAGs) form a functional component of connective tissues that affect the structural and functional integrity of the lower urinary tract (LUT). The specific GAGs of physiological relevance are both nonsulfated (hyaluronan) and sulfated GAGs (chondroitin sulphate [CS], dermatan sulphate [DS], keratan sulphate [KS], and heparan sulphate [HS]). As GAG composition in the LUT is hormonally regulated, we postulated that gonadectomy-induced endocrine imbalance alters the profile of GAGs in the canine LUT. Four regions of the LUT (body and neck of the bladder as well as the proximal and distal urethra) from 20 clinically healthy dogs (5 intact males, 5 intact anoestrus females, 4 castrated males, and 6 spayed females) were collected, wax-embedded and sectioned. Alcian blue staining at critical electrolyte concentrations was performed on the sections to determine total GAGs, hyaluronan, total sulfated GAGs, combined components of CS and DS, as well as KS and HS. The amount of staining was evaluated in 3 tissue layers, i.e., epithelium, subepithelial stroma and muscle within a region. Overall, hyaluronan (67.1%) was the predominant GAG in the LUT. Among sulfated GAGs, a combined component of KS and HS was found to be 61.8% and 38.2% for CS and DS. Gonadal status significantly affected GAG profiles in the LUT (P < 0.01). All GAG components were lower (P < 0.05) in body of the bladder of gonadectomized dogs. Total sulfated GAGs and a combined component of KS and HS were lower (P < 0.05) in all 4 regions of gonadectomized dogs. Except for a combined component of CS and DS, decreases in all GAGs were found more consistently in the muscle compared to other tissue layers. Differences between genders became obvious only when considered along with the effect of gonadal status. In gonadectomized dogs, changes in GAG components in the LUT were more consistent in females compared to males; this may partly explain different levels of risk in the development of urinary incontinence between genders. Quantitative differences in GAG profiles found between intact and gonadectomized dogs indicate a potential role of gonadectomy-induced endocrine imbalance in modifying GAG composition in the canine LUT. Profound alteration in the pattern of GAGs in gonadectomized dogs may compromise structural and functional integrity of the LUT and is possibly involved in the underlying mechanism of urinary incontinence post neutering.

Trastuzumab resistance in breast cancer cell lines: The role of extracellular matrix.

e11135 Background: Trastuzumab is an antibody that blocks the activity of HER2, a member of epidermal growth factor receptor family that is amplified in approximately 20% of breast cancer patients. Trastuzumab significantly improves disease-free and overall survival in these cases. Nevertheless, many patients become resistant to this antibody. Methods: Different human breast cancer cell lines MCF7, SKBR3 and a stable transfected heparanase-1 cDNA MCF7 cells (HPSE-1-MCF7), as well a non tumoral cell line, MCF10A were used for the experimental assays of cell viability, confocal immunofluorescence microscopy and quantitative RT-PCR analysis. Results: Our findings show the role of some extracellular matrix (ECM) components in trastuzumab resistance using breast cancer cell lines assays. MCF7 cells were transfected with heparanase-1 (HPSE-1) cDNA and became completely resistant to trastuzumab. HPSE-1 transfection changes the glycosaminoglycans profile in the transfected MCF7 cells, suggesting that glycosaminoglycans/proteoglycans could be involved in the resistance process. The results showed that trastuzumab binds to heparan sulfate (HS) and this effect is necessary to antibody activity. It was also demonstrated that trastuzumab decreases heparanases isoforms (HPSE-1, HPSE-2), HER2 and the proteoglycan syndecan-1 (Syn-1) mRNA expression in MCF7 cell line, while in MCF7 HPSE-1 transfected cells trastuzumab increases mRNA expression of these molecules. SKBR3 cells presented the highest expression levels of these molecules. The relative proportion of HS chains in Syn-1 core protein seems to influence MCF7 cells sensibility and resistance to this antibody. In addition, HPSE-1/HER2 ratio seems to define trastuzumab resistance, once the relative increase in HPSE-1 expression decrease breast cancer cells sensitivity to trastuzumab. Conclusions: These new insights show the effect of ECM components for trastuzumab efficacy and could be useful when devising strategies for overcoming drug resistance in HER2+ cancers.

Abstract 427: Impact of glycosaminoglycans on lung cancer diagnosis and prognosis of patients

Abstract Introduction: Treatment of lung cancer patients depends on histological type, performance status and clinical stage. Because of limitations of these parameters in predicting disease behavior, different markers have been investigated to better discriminate lung cancer evolution. Glycosaminoglycans(GAGs) are important molecules of the extracellular matrix hydrating and maintaining the osmotic balance of tissues. Many have reported that GAGs have different behaviors when in the presence of malignant tissues. In this study, we sought to examine the sulfated GAGs and hyaluronan (HA) concentrations and their impact on diagnosis and/or prognosis of patients with non small cell lung cancer. Methods: Sulfated GAGs and HA were examined in tumoral and non-tumoral tissues from 45 patients who underwent lobectomy for lung cancer. Their preoperative clinical stages were T1-3N0-1M0 and the mean follow-up was 19.3 months. Their histologic types were adenocarcinomas (n=23), squamous cells carcinomas (SqCC) (n=16) and large cell carcinoma (n=6). Tissue samples were dehydrated with acetone and incubated with a proteolytic enzyme. The HA chains were diluted (1:100) in blocking buffer [0.05M Tris-HCl, pH 7.4, 1% BSA]. The levels of HA were measured by a noncompetitive enzyme-linked immunosorbent assay (ELISA)-like fluorometric assay. The sulfated GAG chains (heparan, dermatan and chondroitin sulfate – HS, DS and CS), were precipitated with ethanol and the pellet was dried and dissolved in DNAse(5μl/mg).The different types of sulfated GAGs and their concentration were identified after gel electrophoresis in diaminopropane buffer. Results: A distinct profile of sulfated GAGs and HA was observed between non-tumoral and tumoral areas. HS and HA showed significantly higher concentration in tumoral than in non-tumoral areas (p=0.02 and p=0.0001, respectively). The adenocarcinomas had higher amounts of HS than SqCC specimens(p=0.02). One hundred % of tumoral areas presented CS while the normal almost never (p&amp;lt;0.0001). The impact of HA was tested on follow-up until death from surgery day. The Kaplan-Meier survival curve showed that tissues with lower concentrations of HA have better long-term survival than those with higher concentrations (Log Rank=3.59; p=0.05). Conclusion: The presence of CS and higher concentrations of HS and HA in patients with lung cancer suggest a possible role of those molecules in this disease, but more importantly provide potential biochemical markers for differentiating normal from lung cancer patients. The correlation between the histologic types and the amounts of HS provide a possible role of this GAG on the development of tumor aggressiveness considering that one of its functions is to bind itself to growth factors and regulate their action. However, further studies are needed to determine whether or not these concentrations are able to be diagnostic/prognostic markers of lung cancer. Financial support: FAPESP Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 427. doi:10.1158/1538-7445.AM2011-427

The chondroitin/dermatan sulfate synthesizing and modifying enzymes in laryngeal cancer: Expressional and epigenetic studies

BackgroundSignificant biochemical changes are observed in glycosaminoglycans in squamous cell laryngeal carcinoma. The most characteristics are in chondroitin/dermatan sulfate fine structure and proportion, which might be due to differential expression of the enzymes involved in their biosynthesis. The aim of the present work was the investigation in expressional and epigenetic level of the enzymes involved in chondroitin/dermatan sulfate biosynthesis in laryngeal cancer.MethodsTissues subjected to total RNA and DNA isolation, and protein extraction. The techniques used in this study were RT-PCR analysis, western blotting and methylation specific PCR.ResultsWe identified that many enzymes were expressed in the cancerous specimens intensively. Dermatan sulfate epimerase was expressed exclusively in the cancerous parts and in minor amounts in healthy tissues; in the macroscopically normal samples it was not detected. Furthermore, chondroitin synthase I and chondroitin polymerizing factor were strongly expressed in the cancerous parts compared to the corresponding normal tissues. Sulfotransferases, like chondroitin 6 sulfotransferase 3, were highly expressed mainly in healthy specimens.ConclusionsThe study of the various chondroitin/dermatan synthesizing enzymes revealed that they were differentially expressed in cancer, in human laryngeal cartilage, leading to specific chondroitin/dermatan structures which contributed to proteoglycan formation with specific features. The expression of the examined enzymes correlated with the glycosaminoglycan profile observed in previous studies.

The composition of hydrogels for cartilage tissue engineering can influence glycosaminoglycan profile

The injectable and hydrophilic nature of hydrogels makes them suitable candidates for cartilage tissue engineering. To date, a wide range of hydrogels have been proposed for articular cartilage regeneration but few studies have quantitatively compared chondrocyte behaviour and extracellular matrix (ECM) synthesis within the hydrogels. Herein we have examined the nature of ECM synthesis by chondrocytes seeded into four hydrogels formed by either temperature change, self-assembly or chemical cross-linking. Bovine articular cartilage chondrocytes were cultured for 14 days in Extracel, Pluronic F127 blended with Type II collagen, Puramatrix and Matrixhyal. The discriminatory and sensitive technique of fluorophore-assisted carbohydrate electrophoresis (FACE) was used to determine the fine detail of the glycosaminoglycans (GAG); hyaluronan and chondroitin sulphate. FACE analysis for chondroitin sulphate and hyaluronan profiles in Puramatrix closely matched that of native cartilage. For each hydrogel, DNA content, viability and morphology were assessed. Total collagen and total sulphated GAG production were measured and normalised to DNA content. Significant differences were found in total collagen synthesis. By day 14, Extracel and Puramatrix had significantly more total collagen than Matrixhyal (1.77+/-0.26 microg and 1.97+/-0.26 microg vs. 0.60+/-0.26 microg; p<0.05). sGAG synthesis occurred in all hydrogels but a significantly higher amount of sGAG was retained within Extracel at days 7 and 14 (p<0.05). In summary, we have shown that the biochemical and biophysical characteristics of each hydrogel directly or indirectly influenced ECM formation. A detailed understanding of the ECM in the development of engineered constructs is an important step in monitoring the success of cartilage regeneration strategies.

Metabolic profile of glycosaminoglycans in bladder and urethra of female rats during and after pregnancy

The objective of this study is to evaluate pregnancy's action on glycosaminoglycans (GAGs) profile in bladder and urethra of female adult rats. Twenty adult female rats were divided into four groups: control, day 10 pregnancy, day 20 pregnancy, and day 5 after delivery. Sulfated GAGs content were determined by densitometry. Hyaluronic acid was assessed by fluorimetric enzyme-linked immunosorbent serologic-like assay. Radioactive GAGs were quantified by measuring radioactivity in electrophoresis gel. Statistical analysis was performed using Kruskal-Wallis and Mann-Whitney tests. In all groups, dermatan sulfate was predominant followed by heparan sulfate and hyaluronic acid. Significant decrease in total GAGs in bladder was observed at the beginning of pregnancy, with progressive increase. Biosynthesis of such molecules showed a similar behavior. In urethra, changes in GAGs content were restricted to the end of pregnancy. Hyaluronic acid content showed a significant increase in bladder during puerperium. We observed different composition in vesical and urethral tissues during pregnancy and in puerperium.

Regulation of Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycan Biosynthesis by 4-Fluoro-glucosamine in Murine Airway Smooth Muscle Cells

The importance of the pathological changes in proteoglycans has driven the need to study and design novel chemical tools to control proteoglycan synthesis. Accordingly, we tested the fluorinated analogue of glucosamine (4-fluoro-N-acetyl-glucosamine (4-F-GlcNAc)) on the synthesis of heparan sulfate (HS) and chondroitin sulfate (CS) by murine airway smooth muscle (ASM) cells in the presence of radiolabeled metabolic precursors. Secreted and cell-associated CS and HS were assessed for changes in size by Superose 6 chromatography. Treatment of ASM cells with 4-F-GlcNAc (100 microM) reduced the quantity (by 64.1-76.6%) and decreased the size of HS/CS glycosaminoglycans associated with the cell layer (K(av) shifted from 0.30 to 0.45). The quantity of CS secreted into the medium decreased by 65.7-73.0%, and the size showed a K(av) shift from 0.30 to 0.50. Treatment of ASM cells with 45 microM and 179 microM 4-F-GlcNAc in the presence of a stimulator of CS synthesis, 4-methylumbelliferyl-beta-D-xyloside, reduced the amount of the xyloside-CS chains by 65.4 and 87.0%, respectively. The size of xyloside-CS chains synthesized in the presence of 4-F-GlcNAc were only slightly larger than those with xyloside treatment alone (K(av) of 0.55 compared with that of 0.6). The effects of 4-F-GlcNAc to inhibit CS synthesis were not observed with equimolar concentrations of glucosamine. We propose that 4-F-GlcNAc inhibits CS synthesis by inhibiting 4-epimerization of UDP-GlcNAc to UDP-GalNAc, thereby depleting one of the substrates required, whereas HS elongation is inhibited by truncation when the nonreducing terminus of the growing chain is capped with 4-F-GlcNAc.

Wild Blueberry (V. angustifolium) Consumption Alters the Composition and Structure of Glycosaminoglycans (GAGs) in Spontaneously Hypertensive Rats (SHR)

The effect of wild blueberries on the composition and structure of GAGs was examined in twenty, 12 week‐old SHRs, fed a control (C) or blueberry‐enriched diet (B) for nine weeks. Thoracic aortae were defatted with methanol/chloroform solution (1:1 v/v), digested with pronase and assayed for total sulfated GAGs and uronic acid content. Aortic specimens from each diet group were pooled together and fractionated on a DEAE‐Sephacel column with a NaCl linear gradient. Identification and characterization of pooled uronic acid‐positive fractions was determined with cellulose acetate electrophoresis, treatment with specific GAG lyases and high performance capillary electrophoresis. A significant decrease in total sulfated GAGs was documented in the SHR‐B, 12.58 ± 0.20 vs. 13.72 ± 0.20 µg/mg aorta in the SHR‐C, p=0.05. Three different GAG populations were identified; hyaluronic acid (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). There was a 25% and 10% increase in HA and HS, respectively, and a 13% decrease in GalAGs in the SHR‐B as compared to SHR‐C group. The changes in the SHR aortic GAG profile produced by wild blueberry consumption may alter cellular signal transduction pathways and subsequently affect the biomechanical milieu of the vascular endothelium.This work was supported by a USDA/CSREES (WBANA) Grant and a Greek State Scholarship Foundation Fellowship (IKY).Grant Funding SourceWBANA, IKY

Depth‐dependent profiles of glycosaminoglycans in articular cartilage by μMRI and histochemistry

To quantify the distribution profile of glycosaminoglycans (GAGs) in articular cartilage with the magnetic resonance imaging (MRI) gadolinium (Gd) contrast method (the dGEMRIC procedure in clinical MRI) and correlate with histochemical results. Fresh canine cartilage from seven humeral heads was harvested. Sixteen cartilage specimens were imaged at 13 microm pixel resolution using the microMRI T1-Gd method to generate 2D GAG maps in cartilage. Nineteen cartilage specimens from adjacent locations on the same joints were papain-digested to quantify the bulk GAG content in tissue. In addition, six cartilage specimens were microtomed into 40-microm serial sections that were parallel with the articular surface. These sections were biochemically analyzed individually to determine the depth-dependent profiles of GAG concentration. The GAG concentrations between the microMRI measurement and the bulk biochemical method have statistically significant agreement. The depth-dependent GAG profiles from the histochemical method (40 microm depth resolution) have similar line shapes as that determined by microMRI at 13 microm resolution. The GAG concentration as measured by microMRI T1-Gd contrast method provides an accurate account of the macromolecular content in articular cartilage.

Reduced supportive capacity of bone marrow stroma upon chemotherapy is mediated via changes in glycosaminoglycan profile

High dose chemotherapy and radiation have been found to impair the hematopoiesis-supportive capacity of bone marrow stroma. We now provide evidence for an important role of chemotherapy-induced alterations in stromal glycosaminoglycans (GAGs) in reduction of the supportive properties of stromal fibroblasts. Exposure to cytarabine resulted in a pronounced increase in hyaluronan, both in the cell/matrix ( p < 0.03) and supernatant fraction ( p < 0.05). Gene expression analysis showed a corresponding increase in gene expression of hyaluronan synthase 1, indicating that the increase in hyaluronan is at least partly under genetic control. Functionally, hyaluronan significantly inhibited the proliferation of early megakaryocytic progenitor cells in a dose dependent way ( p = 0.01). The increase in hyaluronan was confirmed in vivo by showing a > 2-fold increase in bone marrow hyaluronan of patients after chemo- and/or radiotherapy as conditioning for an allogeneic stem cell transplantation, indicating physiologically relevance. Furthermore, there was a trend towards a decrease in the amount and sulfation of stromal heparan sulfate proteoglycans upon exposure to cytarabine, resulting in a 40% reduced binding of SDF1-α to stromal cells ( p < 0.05). In conclusion, there is a pronounced effect of cytarabine treatment on the expression of genes involved in GAG synthesis and degradation, affecting the synthesis and function of stromal GAGs. Our results indicate that chemotherapy-induced changes in stromal GAG profile are likely to affect normal hematopoiesis.

Glycosaminoglycan profiles of repair tissue formed following autologous chondrocyte implantation differ from control cartilage

Currently, autologous chondrocyte implantation (ACI) is the most commonly used cell-based therapy for the treatment of isolated femoral condyle lesions of the knee. A small number of centres performing ACI have reported encouraging long-term clinical results, but there is currently a lack of quantitative and qualitative biochemical data regarding the nature of the repair tissue. Glycosaminoglycan (GAG) structure influences physiological function and is likely to be important in the long-term stability of the repair tissue. The objective of this study was to use fluorophore-assisted carbohydrate electrophoresis (FACE) to both quantitatively and qualitatively analyse the GAG composition of repair tissue biopsies and compare them with age-matched cadaveric controls. We used immunohistochemistry to provide a baseline reference for comparison. Biopsies were taken from eight patients (22 to 52 years old) 1 year after ACI treatment and from four cadavers (20 to 50 years old). FACE quantitatively profiled the GAGs in as little as 5 μg of cartilage. The pattern and intensity of immunostaining were generally comparable with the data obtained with FACE. In the ACI repair tissue, there was a twofold reduction in chondroitin sulphate and keratan sulphate compared with age-matched control cartilage. By contrast, there was an increase in hyaluronan with significantly shorter chondroitin sulphate chains and less chondroitin 6-sulphate in repair tissue than control cartilage. The composition of the repair tissue thus is not identical to mature articular cartilage.

Uronate Peaks and Urinary Hyaluronic Acid Levels Correlate With Interstitial Cystitis Severity

Levels of uronate, a basic component of urothelial glycosaminoglycans, are increased in urine specimens of patients with interstitial cystitis with severe symptoms. In this study we examined the urinary glycosaminoglycan profile and correlated the profile and urinary hyaluronic acid (a glycosaminoglycan) levels with symptom severity. Urine specimens and completed O'Leary-Sant interstitial cystitis symptom and problem indexes questionnaires were obtained from 29 patients with interstitial cystitis, 14 normal individuals, and 14 patients with other benign pelvic and bladder conditions. Patients with interstitial cystitis were divided into group 1-1 or both indexes less than 50% maximum score, and group 2-both indexes 50% of maximum score or greater. All patients met the National Institutes of Diabetes and Digestive and Kidney Diseases criteria except regarding glomerulation. In a followup study 30 urine specimens were collected from 8 patients with interstitial cystitis and from 4 normal individuals during 12 months. The urinary glycosaminoglycan profile was determined by gel filtration chromatography. Glycosaminoglycan peaks were analyzed by polyacrylamide gel electrophoresis. Urinary hyaluronic acid levels were determined by the hyaluronic acid test. Group 2 urine specimens contained 3 uronate peaks, whereas urine specimens from normal individuals and patients in group 1 contained 1 or 2 peaks. Peak 1 consisted of macromolecular glycosaminoglycans whereas peaks 2 and 3 contained oligosaccharides. Urinary hyaluronic acid levels were 3 to 4-fold increased in group 2. Glycosaminoglycan profile and hyaluronic acid levels detected interstitial cystitis severity with 83% sensitivity, and 89.7% and 74.4% specificity, respectively. Interstitial cystitis urothelial cells/tissues also over expressed hyaluronic acid synthase 1 (which synthesizes hyaluronic acid) compared to normal urothelial cells/tissues. In the followup study urinary uronate levels, glycosaminoglycan profile and hyaluronic acid levels detected patients with severe symptoms with 73% sensitivity and 87% to 94% specificity. In both studies uronate, glycosaminoglycan profile and hyaluronic acid levels significantly correlated with interstitial cystitis severity (p <0.001). Urinary glycosaminoglycan profile, uronate content and hyaluronic acid levels are potentially useful markers for monitoring interstitial cystitis severity, and are likely to be involved in interstitial cystitis pathophysiology.

Effect of bitter gourd and spent turmeric on constituents of glycosaminoglycans in different tissues in streptozotocin induced diabetic rats

Diet is now one of the well established means in the management of diabetes. Bitter gourd and spent turmeric at 10% level were tested for their efficacy on glycosaminoglycan metabolism in various tissues viz., liver, spleen, lungs, heart and testis in control, diabetic and treated rats. The glycosaminoglycans (GAGs) were isolated from defatted and dried tissues. The contents of sulfated GAGs decreased in all the tissues and the decrease was more prominent in heart and testis. In the isolated GAGs, contents of total sugar, amino sugar, uronic acid and sulfate were studied. Decrease in total sugar content was maximum in testis. Amino sugar content decreased considerably in testis (38%) and lungs (15%). The content of uronic acid also decreased in testis (33%) besides heart (29%) and liver (25%). Sulfate groups in GAGs perform pivotal functions in many biological events and decrease in sulfate content was significant in heart (40%), testis (37%) and liver (37%). GAGs profile on the cellulose acetate electrophoresis revealed that heparan sulfate (HS), hyaluronic acid (HA) and chondroitin sulfate/dermatan sulfate (CS/DS) were present in liver, spleen and lungs. HS, CS were present in heart, DS/CS was observed in testis. The observed beneficial effects in GAGs metabolism during diabetes may be due to the presence of high amounts of dietary fibres present in bitter gourd and spent turmeric, besides, possible presence of bioactive compounds in one or both of them.

Alterations in serum glycosaminoglycan profiles in Graves' patients

Qualitative and quantitative analyses of glycosaminoglycans (GAGs) in serum obtained from Graves' disease (GD) patients without extrathyroidal complications were carried out to provide a clearer understanding of the role of these macromolecules in the disease pathogenesis. GAGs were isolated from-SIMEL (Italian Society of Laboratory Medicine) Inter-associative Study Group on Diabetes Mellitus serum of 17 GD patients before treatment and after attainment of the euthyroid state, as well as from 20 healthy individuals. GAGs were quantified using the hexuronic acid assay and subjected to electrophoretic fractionation. Increased amounts of total GAGs were found in GD patients. Attainment of euthyroidism led to a decrease in, but not normalization of, total serum GAGs level. Electrophoretic analyses of GAGs before and after treatment identified the presence of chondroitin sulfate (CS), heparan sulfate/heparin (HS/H) and dermatan sulfate (DS) in serum from healthy subjects and GD patients. CS was the predominant serum GAG constituent in all subjects investigated. Enhanced CS levels in both GD patient groups were accompanied by increased structural heterogeneity of these compounds. Normalization of thyroid function did not change CS levels. DS levels in serum of untreated GD patients were elevated in comparison to healthy subjects. Anti-thyroid treatment led to a significant decrease in DS to levels below those in controls. DS in all serum samples investigated displayed a similar structure. HS/H levels in serum of untreated GD patients was seven-fold higher than in healthy subjects. In addition, HS/H in untreated GD patients were characterized by higher structural heterogeneity than those isolated from control subjects and euthyroid GD patients. Anti-thyroid therapy led to a decrease in HS/H concentrations towards normal values. Our results indicate that in the course of GD, the metabolism of particular types of GAGs is regulated by different mechanisms, including a hyperthyroid state and immunological abnormalities. Furthermore, qualitative and quantitative changes in serum GAGs seem to reflect GD-associated systemic changes in extracellular matrix properties.