Glycosaminoglycan Profile Research Articles

Remodeling of Glycosaminoglycans During Differentiation of Adult Human Bone Mesenchymal Stromal Cells Toward Hepatocytes.

There is a critical need to generate functional hepatocytes to aid in liver repair and regeneration upon availability of a renewable, and potentially personalized, source of human hepatocytes (hHEP). Currently, the vast majority of primary hHEP are obtained from human tissue through cadavers. Recent advances in stem cell differentiation have opened up the possibility to obtain fully functional hepatocytes from embryonic or induced pluripotent stem cells, or adult stem cells. With respect to the latter, human bone marrow mesenchymal stromal cells (hBMSCs) can serve as a source of autogenetic and allogenic multipotent stem cells for liver repair and regeneration. A major aspect of hBMSC differentiation is the extracellular matrix (ECM) composition and, in particular, the role of glycosaminoglycans (GAGs) in the differentiation process. In this study, we examine the influence of four distinct culture conditions/protocols (T1-T4) on GAG composition and hepatic markers. α-Fetoprotein and hepatocyte nuclear factor-4α were expressed continually over 21 days of differentiation, as indicated by real time quantitative PCR analysis, while albumin (ALB) expression did not begin until day 21. Hepatocyte growth factor (HGF) appears to be more effective than activin A in promoting hepatic-like cells through the mesenchymal-epithelial transition, perhaps due to the former binding to the HGF receptor to form a unique complex that diversifies the biological functions of HGF. Of the four protocols tested, uniform hepatocyte-like morphological changes, ALB secretion, and glycogen storage were found to be highest with protocol T2, which involves both early- and late-stage combinations of growth factors. The total GAG profile of the hBMSC ECM is rich in heparan sulfate (HS) and hyaluronan, both of which fluctuate during differentiation. The GAG profile of primary hHEP showed an HS-rich ECM, and thus, it may be possible to guide hBMSC differentiation to more mature hepatocytes by controlling the GAG profile expressed by differentiating cells.

MP72-19 PLASMA GLYCOSAMINOGLYCAN SCORES IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II1 Apr 2018MP72-19 PLASMA GLYCOSAMINOGLYCAN SCORES IN RENAL CELL CARCINOMA Kyle A. Blum, Francesco Gatto, Mazyar Ghannat, Alejandro Sanchez, Francesca Maccari, Fabio Galeotti, James Hsieh, Nicola Volpi, A. Ari Hakimi, and Jens Nielsen Kyle A. BlumKyle A. Blum More articles by this author , Francesco GattoFrancesco Gatto More articles by this author , Mazyar GhannatMazyar Ghannat More articles by this author , Alejandro SanchezAlejandro Sanchez More articles by this author , Francesca MaccariFrancesca Maccari More articles by this author , Fabio GaleottiFabio Galeotti More articles by this author , James HsiehJames Hsieh More articles by this author , Nicola VolpiNicola Volpi More articles by this author , A. Ari HakimiA. Ari Hakimi More articles by this author , and Jens NielsenJens Nielsen More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2303AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Glycosaminoglycan (GAG) levels are measurably altered in the plasma of patients with clear cell renal cell carcinoma (ccRCC). GAG scores have been used to detect ccRCC in a cohort of patients with metastatic disease with 92.7% accuracy (Gatto et al, Cell Reports, 2016). However, it is unknown if GAG scores can detect RCC in earlier stages or non-ccRCC histologies. METHODS In this retrospective study, pre-operative plasma GAG levels from 162 RCC patients were compared to 19 healthy controls between 5/2011-2/2014. GAG scores were generated using 19 pre-specified properties and measured using capillary electrophoresis with laser induced fluorescence. GAG profile differences in RCC versus healthy controls were assessed using unsupervised clustering methods. A discovery set of 68 RCC vs. 19 healthy samples were first analyzed to update the historical GAG score. The new GAG score accuracy in RCC detection versus healthy subjects was validated using the remaining 94 RCC samples. RESULTS Median age was similar between RCC and healthy cohorts, 60 years (IQR: 52-67) vs. 55 years (IQR: 50-60), respectively. In the RCC cohort, 113 (70%) were ccRCC and 49 (30%) non-ccRCC. RCC stage included 86 (53%) pT1, 66 (41%) pT2-3, and 12 (7%) pT4. GAG profiles in RCC clustered separately from healthy volunteers (Figure 1A). In the first discovery set (n=68), the GAG score distinguished RCC from healthy subjects with an AUC equal to 0.999, 94.7% specificity and 100% sensitivity at an optimal cut-off equal to 0.87 (Figure 1B). In the validation set (n=94), the GAG score achieved an AUC equal to 0.994 (95% CI: 0.985 - 1) and 95.7% sensitivity (Figure 1B). GAG scores did not correlate with age or gender. GAG scores were elevated in all RCC samples compared to normal controls, irrespective and uncorrelated to stage, grade or histology. CONCLUSIONS Plasma GAG scores are measurably elevated in RCC compared to healthy individuals. It was possible to detect RCC irrespective of stage or histology with 95.7% sensitivity. GAG scores did not correlate with pathologic stage, grade, or histology. These findings suggest that GAG alterations occur early in tumor formation but are likely independent of progression. These findings warrant prospective validation to assess the clinical utility of pre-operative GAG scores as biomarkers for RCC. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e959-e960 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Kyle A. Blum More articles by this author Francesco Gatto More articles by this author Mazyar Ghannat More articles by this author Alejandro Sanchez More articles by this author Francesca Maccari More articles by this author Fabio Galeotti More articles by this author James Hsieh More articles by this author Nicola Volpi More articles by this author A. Ari Hakimi More articles by this author Jens Nielsen More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A method for characterising human intervertebral disc glycosaminoglycan disaccharides using liquid chromatography-mass spectrometry with multiple reaction monitoring.

Intervertebral disc (IVD) degeneration results in the depletion of proteoglycans and glycosaminoglycans (GAGs), which can lead to structural and mechanical loss of IVD function, ingrowth of nociceptive nerve fbres and eventually discogenic pain. Specifc GAG types as well as their disaccharide paterns can be predictive of disease and degeneration in several tissues but have not been comprehensively studied within the IVD. A highly sensitive mass spectrometry based technique with multiple reaction monitoring (MRM) was used to provide characterisation of chondroitin sulphate (CS), hyaluronic acid (HA), heparan sulphate (HS) and their disaccharide sulphation paterns across diferent anatomical regions of human IVDs. Principal component analysis further distinguished important regional variations and proposed potential ageing variations in GAG profles. CS was the GAG in greatest abundance in the IVD followed by HA and HS. Principal component analysis identifIed clear separation of GAG profiles between nucleus pulposus and annulus fibrosus in young and old specimens. Distinct patterns of predominantly expressed disaccharides of CS and HS between young and old IVD samples, provided preliminary evidence that important alterations in disaccharides occur within IVDs during ageing. This technique offered a novel approach to identify and quantify specific GAG disaccharides in human IVDs and the data presented were the first to offer insight into the spatial distribution as well as association with ageing of GAGs and GAG disaccharide sulphation patterns across the human IVD.

The influence of hyperglycemia on the remodeling of urethral connective tissue in pregnant rats

ObjectiveTo analyze the distribution and quantification of the key structural extracellular matrix components of the urethral tissue in a rat model of hyperglycemia and pregnancy. Study designA total of 120 female Wistar rats were distributed into the following four experimental groups: virgin, pregnant, hyperglycemic and hyperglycemic + pregnant groups. The urethra was harvested for histochemical, morphometric, immunohistochemical, Western blot and glycosaminoglycan analyses. All protocols were approved by the Institutional Animal Care and Use Committee of Botucatu Medical School (process number 828–2010). ResultsThe hyperglycemic + pregnant group showed significantly increased stiffness in urethral tissue. The total striated muscle was decreased, with increased deposition of collagen fibers around the muscle fibers and a change in the organization of the collagen fibrils. An increase in the relative collagen type I/III ratio and a decrease in total glycosaminoglycans were also observed. ConclusionsThis study provides the first line of experimental evidence supporting a metabolic relationship between hyperglycemia and urethral remodeling of connective tissue in pregnant rats. The different organization of the collagen fibrils and the profile of glycosaminoglycans found in urethral samples suggest that the pathology of the urethral fibromuscular system could be related to hyperglycemia-induced pelvic floor dysfunction in women, which has direct clinical implications with the possibility to develop new multidisciplinary treatments for improving the health care of these women.

Glycosaminoglycan and Proteoglycan-Based Biomaterials: Current Trends and Future Perspectives.

Proteoglycans and their glycosaminoglycans (GAG) are essential for life as they are responsible for orchestrating many essential functions in development and tissue homeostasis, including biophysical properties and roles in cell signaling and extracellular matrix assembly. In an attempt to capture these biological functions, a range of biomaterials are designed to incorporate off-the-shelf GAGs, typically isolated from animal sources, for tissue engineering, drug delivery, and regenerative medicine applications. All GAGs, with the exception of hyaluronan, are present in the body covalently coupled to the protein core of proteoglycans, yet the incorporation of proteoglycans into biomaterials remains relatively unexplored. Proteoglycan-based biomaterials are more likely to recapitulate the unique, tissue-specific GAG profiles and native GAG presentation in human tissues. The protein core offers additional biological functionality, including cell, growth factor, and extracellular matrix binding domains, as well as sites for protein immobilization chemistries. Finally, proteoglycans can be recombinantly expressed in mammalian cells and thus offer genetic manipulation and metabolic engineering opportunities for control over the protein and GAG structures and functions. This Progress Report summarizes current developments in GAG-based biomaterials and presents emerging research and future opportunities for the development of biomaterials that incorporate GAGs presented in their native proteoglycan form.

Extracellular matrix alterations in the Peyronie’s disease

Peyronie’s disease is characterized by fibrous plaque formation of the tunica albuginea, causing penile deformity and fertility problems. The aim of the present study was to investigate alterations in the extracellular matrix in Peyronie’s disease. The study used tissues collected by surgical procedure from individuals that presented a well-established disease, while control samples were obtained by biopsies of fresh cadavers. Immunohistochemistry analysis followed by digital quantification was performed to evaluate TGF-β, heparanases and metalloproteinases (MMPs). The profile of sulfated glycosaminoglycans, chondroitin sulfate and dermatan sulfate was determined by agarose gel electrophoresis, while hyaluronic acid quantification was obtained by an ELISA-like assay. The expression of mRNA was investigated for syndecan-1 proteoglycan (Syn-1), interleukine-6 (IL-6), hyaluronic acid synthases, and hyaluronidases. Pathologic features showed decreased apoptosis and blood vessel number in Peyronie’s tissues. TGF-β and IL-6 were significantly enhanced in Peyronie’s disease. There was an increased expression of heparanases, though no alteration was observed for MMPs. Hyaluronic acid as well as hyaluronic acid synthases, hyaluronidases, and dermatan sulfate were not changed, while the level of chondroitin sulfate was significantly (P=0.008, Mann-Whitney test) increased in Peyronie’s samples. Heparanases and sulfated glycosaminoglycans seem to be involved in extracellular matrix alterations in Peyronie’s disease.

A case study: Glycosaminoglycan profiles of autologous chondrocyte implantation (ACI) tissue improve as the tissue matures

BackgroundAutologous chondrocyte implantation (ACI) has been used to treat cartilage defects in thousands of patients worldwide with good clinical effectiveness 10–20years after implantation. Information concerning the quality of the repair cartilage is still limited because biopsies are small and rare. Glycosaminoglycan structure influences physiological function and is likely to be important in the long term stability of the repair tissue. The aim of this study was to assess glycosaminoglycans in ACI tissue over a two year period. MethodsBiopsies were taken from one patient (25years old) at 12months and 20months post-ACI-treatment and from three normal cadavers (21, 22 and 25years old). Fluorophore-assisted carbohydrate electrophoresis (FACE) was used to quantitatively assess the individual glycosaminoglycans. ResultsAt 12months the ACI biopsy had 40% less hyaluronan than the age-matched cadaveric biopsies but by 20months the ACI biopsy had the same amount of hyaluronan as the controls. Both the 12 and 20month ACI biopsies had less chondroitin sulphate disaccharides and shorter chondroitin sulphate chains than the age-matched cadaveric biopsies. However, chondroitin sulphate chain length doubled as the ACI repair tissue matured at 12months (3913Da±464) and 20months (6923Da±711) and there was less keratan sulphate as compared to the controls. ConclusionsAlthough the glycosaminoglycan composition of the repair tissue is not identical to mature articular cartilage its quality continues to improve with time.

Expression of heparanase in basal cell carcinoma and squamous cell carcinoma.

BackgroundHeparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer.ObjectivesEvaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control).MethodsGlycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR).ResultsThe A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma.ConclusionThe glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.

UPLC-MS/MS detection of disaccharides derived from glycosaminoglycans as biomarkers of mucopolysaccharidoses

Mucopolysaccharidoses (MPSs) are a group of disorders resulting from primary defects in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). Depending on the specific enzyme defect, the catabolism of one or more GAGs is blocked leading to accumulation in tissues and biological fluids. GAG measurements are important for high-risk screening, diagnosis, monitoring treatment efficacy, and patient follow up. The dimethylmethylene blue (DMB) spectrophotometric method commonly used in most biochemical genetics laboratories relies on a non-specific total GAG analysis which has led to false positive results, and even false negative results (mainly for MPS III and IV patients). The main objective of our project was to devise and validate a reliable tandem mass spectrometry multiplex analysis for the urine quantitation of four GAGs (dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and chondroitin sulfate (CS)) for an eventual technological transfer to the clinic. The developed methodology is rapid (7 min) and our results showed good intraday and interday precision (RSDs ≤ 8.7%) and accuracy (Biases range: −12.0%–18.4%). Linearity was good (r2 > 0.995) for DS, HS, CS, and KS calibration curves. In comparison with the DMB spectrophotometric method, this multiplex tandem mass spectrometry method allows GAG fractionation, thus a differentiation of MPS types, except for MPS I and II which are characterized by the same GAG profile. The devised method is a useful and reliable tool for diagnosis of MPS patients, as well as their monitoring and follow up, as shown by longitudinal studies.

Glycosaminoglycan Profiling in Patients' Plasma and Urine Predicts the Occurrence of Metastatic Clear Cell Renal Cell Carcinoma.

Metabolic reprogramming is a hallmark of clear cell renal cell carcinoma (ccRCC) progression. Here, we used genome-scale metabolic modeling to elucidate metabolic reprogramming in 481 ccRCC samples and discovered strongly coordinated regulation of glycosaminoglycan (GAG) biosynthesis at the transcript and protein levels. Extracellular GAGs are implicated in metastasis, so we speculated that such regulation might translate into a non-invasive biomarker for metastatic ccRCC (mccRCC). We measured 18 GAG properties in 34 mccRCC samples versus 16 healthy plasma and/or urine samples. The GAG profiles were distinctively altered in mccRCC. We derived three GAG scores that distinguished mccRCC patients with 93.1%-100% accuracy. We validated the score accuracies in an independent cohort (up to 18 mccRCC versus nine healthy) and verified that the scores normalized in eight patients with no evidence of disease. In conclusion, coordinated regulation of GAG biosynthesis occurs in ccRCC, and non-invasive GAG profiling is suitable for mccRCC diagnosis.

Metabolism of glycosaminoglycans in the course of juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) is a non-homogeneous autoimmune children's disease which, despite the applied therapy, has a progressive character with recurrences, leading to damage of joint structures. Progressive wearing of the joint cartilage in the course of JIA, which results from the imbalance between the biological strength of the cartilage, its function and exerted pressure forces, is linked to metabolic disorders of extracellular matrix (ECM) components. Among the latter compounds, the proteoglycan (PG) aggrecan plays a particular role in maintaining the mechanical-immunological properties of the cartilage. These functions are directly related to chains of glycosaminoglycans (GAGs), covalently linked to the core protein of PGs. Therefore, every change of GAGs metabolism linked to an increase of the rate of degradation or with a decrease of their biosynthesis may have pathological consequences. In this paper we aim to describe plausible mechanisms leading to observed disorders of aggrecan transformation in children, which are reflected in the profile of plasma GAGs. Therefore, we describe the plausible role of factors related to catabolism and synthesis of PGs/GAGs as well as the contribution of immunological processes to shaping the changes of extracellular matrix components in the course of JIA.

Managing chronic bladder diseases with the administration of exogenous glycosaminoglycans: an update on the evidence.

Although the pathophysiology of acute chronic cystitis and other 'sensory' disorders, i.e. painful bladder syndrome (PBS) or interstitial cystitis (IC), often remains multifactorial, there is a wide consensus that such clinical conditions may arise from a primary defective urothelium lining or from damaged glycosaminoglycans (GAGs). A 'cascade' of events starting from GAG injury, which fails to heal, may lead to chronic bladder epithelial damage and neurogenic inflammation. To restore the GAG layer is becoming the main aim of new therapies for the treatment of chronic cystitis and PBS/IC. Preliminary experiences with GAG replenishment for different pathological conditions involving the lower urinary tract have been reported. There is a range of commercially available intravesical formulations of these components, alone or in combination. Literature evidence shows that exogenous intravesical hyaluronic acid markedly reduces recurrences of urinary tract infections (UTIs). Patients treated with exogenous GAGs have fewer UTI recurrences, a longer time to recurrence and a greater improvement in quality of life. Exogenous intravesical GAGs have been used for the treatment of PBS/IC. Despite the limitations of most of the studies, findings confirmed the role of combination therapy with hyaluronic acid and chondroitin sulfate as a safe and effective option for the treatment of PBS/IC. To prevent and/or treat radiotherapy and chemotherapy induced cystitis, GAG replenishment therapy has been used showing preliminary encouraging results. The safety profile of exogenous GAGs has been reported to be very favourable, without adverse events of particular significance.

Glycosaminoglycans affect heparanase location in CHO cell lines.

Glycosaminoglycans (GAG) play a ubiquitous role in tissues and cells. In eukaryotic cells, heparan sulfate (HS) is initially degraded by an endo-β-glucuronidase called heparanase-1 (HPSE). HS oligosaccharides generated by the action of HPSE intensify the activity of signaling molecules, activating inflammatory response, tumor metastasis, and angiogenesis. The aim of the present study was to understand if sulfated GAG could modulate HPSE, since the mechanisms that regulate HPSE have not been completely defined. CHO-K1 cells were treated with 4-methylumbelliferone (4-MU) and sodium chlorate, to promote total inhibition of GAG synthesis, and reduce the sulfation pattern, respectively. The GAG profile of the wild CHO-K1 cells and CHO-745, deficient in xylosyltransferase, was determined after [(35)S]-sulfate labeling. HPSE expression was determined via real-time quantitative polymerase chain reaction. Total ablation of GAG with 4-MU in CHO-K1 inhibited HPSE expression, while the lack of sulfation had no effect. Interestingly, 4-MU had no effect in CHO-745 cells for these assays. In addition, a different enzyme location was observed in CHO-K1 wild-type cells, which presents HPSE mainly in the extracellular matrix, in comparison with the CHO-745 mutant cells, which is found in the cytoplasm. In view of our results, we can conclude that GAG are essential modulators of HPSE expression and location. Therefore, GAG profile could impact cell behavior mediated by the regulation of HPSE.

Composition and significance of glycosaminoglycans in the uterus and placenta of mammals

Glycosaminoglycans (GAGs) are heteropolysaccharides in mammalian tissue and consist of repeated disaccharide units with mono-sulfated or non-sulfated monosaccharides. GAGs are important components of the Extracellular Matrix (ECM) with several physiological roles, in the recognition, migration, adhesion, proliferation and differentiation processes. They are also important in angiogenesis, blood homeostasis, immune reactions, follicule development and also in the development of pathologies such as infertility, tumors and metastases. It has been shown that the profile of glycosaminoglycans in the uterine and placental tissues is highly variable throughout the reproductive cycle and during pregnancy. It may be directly related to their physiological or pathological functions in the tissue. The latter has recently triggered special clinical interest. Current review collaborates for a deeper knowledge on the profile and importance of GAGs in uterine and placental tissues throughout the reproductive cycle and pregnancy. It also covers information on the involvement of these molecules in pathological processes.

Alterations of plasma glycosaminoglycan profile in patients with rheumatoid arthritis in relation to disease activity

BackgroundQualitative and quantitative evaluation of plasma glycosaminoglycans (GAGs) of rheumatoid arthritis (RA) patients in relation to disease activity estimated by DAS28 score was evaluated. MethodsGAGs were quantified by hexuronic acid assay and electrophoretic fractionation. Keratan sulfate (KS) and hyaluronic acid (HA) were measured by immunoassay. ResultsChondroitin/dermatan sulfate (CS/DS) and heparan sulfate/heparin (HS/H) in plasma of healthy subjects and RA patients were stated. Total GAGs, CS, HS/H and HA levels were higher in patients with high and moderate disease activity than in controls. Total GAGs and CS levels in patients with high disease activity were elevated in comparison to patients with low disease activity. HS/H levels in patients with high and moderate activity were elevated in comparison to those with low disease activity. KS levels were increased in all patient groups in comparison to controls. Total GAGs, CS, HS/H and HA levels were positively correlated with DAS28 and CRP. ConclusionsStructural tissue damage/remodeling of the extracellular matrix occurs in RA, which is reflected in the qualitative and quantitative changes of plasma GAGs. The above changes depend on DAS28 and may contribute to systemic changes in the properties of the extracellular matrix.

Modulation of heparan sulfate biosynthesis by sodium butyrate in recombinant CHO cells

Sodium butyrate, a histone deacetylase inhibitor, has been used to improve transgene expression in Chinese hamster ovary (CHO) cells. The current study explores the impact of butyrate treatment on heparan sulfate (HS) biosynthesis and structural composition in a recombinant CHO-S cell line expressing enzymes in the heparin (HP)/(HS) biosynthetic pathway (Dual-10 stably expressing NDST2 and HS3st1). Flow cytometric analysis showed that antithrombin binding was increased in Dual-10 cells and basic fibroblast growth factor binding was decreased in response to sodium butyrate treatment. The results were in agreement with the AMAC-LCMS (2-aminoacridine-tagged HS/HP analysis by liquid chromatography mass spectrometry) data that showed that there was an increase in heparan sulfate tri-sulfated disaccharides and a decrease in N-sulfated disaccharides in the butyrate-treated cells. However, we could not detect any changes in the chondroitin sulfate pathway in Dual-10 cells treated with butyrate. The current study is the first to report the effect of butyrate on glycosaminoglycan profiles.

THE ANTICOAGULANT ACTIVITY AND STRUCTURAL CHARACTERIZATION OF FRACTIONATED AND PURIFIED GLYCOSAMINOGLYCANS FROM VENERID CLAM MERETRIX CASTA (CHEMNITZ)

Heparin (heterogeneous anionic polysaccharide), the glycosaminoglycans (GAGs) has been used as an anticoagulant for several years in clinical studies. In the present study, GAGs from Meretrix casta were fractionated by ion exchange chromatography (Amberlite IRA-900). The active fractions were identified by agarose gel electrophoresis and metachromatic assay (confirmatory test) pooled, dialyzed, and purified by barium acetate. The molecular weights of both fractionated and purified GAGs were observed through polyacrylamide gradient gel electrophoresis and its structural characterization relied on Fourier transform-infrared and 1H-NMR spectroscopy. Finally the activated partial thromboplastin time and the disaccharides profile of GAGs were also determined. The outcome of results of this study suggested that the extracted GAGs from M. casta could be an alternative source of heparin.

Collagen and Glycosaminoglycan Profiles in the Canine Cervix during Different Stages of the Estrous Cycle and in Open- and Closed-Cervix Pyometra

ABSTRACTThe extracellular matrix of the cervix that comprises collagen, elastin, proteoglycans and glycosaminoglycans (GAGs) is thought to have an essential role in cervical relaxation. This study investigated the proportion of collagen and smooth muscle as well as the GAGs in cervices obtained from healthy bitches at different stages of the estrous cycle and bitches with open- and closed-cervix pyometra. Cervices were collected after ovariohysterectomy. The proportion of collagen to smooth muscle was determined using Masson’s trichrome staining. Alcian blue staining was used to evaluate the relative distribution of cervical GAGs. The proportion of cervical collagen relative to smooth muscle was higher at estrus compared to anestrus (P≤0.05). It was also higher (P≤0.05) in bitches with open- compared to those with closed-cervix pyometra. Overall, hyaluronan (HA) was the predominant GAG in the canine cervix. In the luminal epithelium, the staining intensity for HA was stronger in estrus than in anestrus (P≤0.05), but not in diestrus (P>0.05). On the contrary, the intensity for the combined keratan sulfate (KS) and heparan sulfate (HS) was stronger in anestrus than in estrus and diestrus (P≤0.05). In bitches with pyometra, the staining intensity of the stroma for KS and HS was weaker in open- compared to closed-cervix pyometra (P≤0.05). Collectively, the different profiles of collagen and GAG suggest that the metabolism of both collagen and GAGs in the canine cervix is associated with hormonal statuses during the estrous cycle and cervical patency of bitches with pathological uterine conditions, such as pyometra.

Heparan sulfate mediates trastuzumab effect in breast cancer cells

BackgroundTrastuzumab is an antibody widely used in the treatment of breast cancer cases that test positive for the human epidermal growth factor receptor 2 (HER2). Many patients, however, become resistant to this antibody, whose resistance has become a major focus in breast cancer research. But despite this interest, there are still no reliable markers that can be used to identify resistant patients. A possible role of several extracellular matrix (ECM) components—heparan sulfate (HS), Syn-1(Syndecan-1) and heparanase (HPSE1)—in light of the influence of ECM alterations on the action of several compounds on the cells and cancer development, was therefore investigated in breast cancer cell resistance to trastuzumab.MethodsThe cDNA of the enzyme responsible for cleaving HS chains from proteoglycans, HPSE1, was cloned in the pEGFP-N1 plasmid and transfected into a breast cancer cell lineage. We evaluated cell viability after trastuzumab treatment using different breast cancer cell lines. Trastuzumab and HS interaction was investigated by confocal microscopy and Fluorescence Resonance Energy Transfer (FRET). The profile of sulfated glycosaminoglycans was also investigated by [35S]-sulfate incorporation. Quantitative RT-PCR and immunofluorescence were used to evaluate HPSE1, HER2 and Syn-1 mRNA expression. HPSE1 enzymatic activity was performed using biotinylated heparan sulfate.ResultsBreast cancer cell lines responsive to trastuzumab present higher amounts of HER2, Syn-1 and HS on the cell surface, but lower levels of secreted HS. Trastuzumab and HS interaction was proven by FRET analysis. The addition of anti-HS to the cells or heparin to the culture medium induced resistance to trastuzumab in breast cancer cells previously sensitive to this monoclonal antibody. Breast cancer cells transfected with HPSE1 became resistant to trastuzumab, showing lower levels of HER2, Syn-1 and HS on the cell surface. In addition, HS shedding was increased significantly in these resistant cells.ConclusionTrastuzumab action is dependent on the availability of heparan sulfate on the surface of breast cancer cells. Furthermore, our data suggest that high levels of heparan sulfate shed to the medium are able to capture trastuzumab, blocking the antibody action mediated by HER2. In addition to HER2 levels, heparan sulfate synthesis and shedding determine breast cancer cell susceptibility to trastuzumab.

RELATIONSHIP BETWEEN BIOCHEMICAL PARAMETERS OF THE SPINAL MOTION SEGMENT AND OUTCOME OF SURGERY FOR HERNIATED LUMBAR INTERVERTEBRAL DISC

Objective. To analyze natural biochemical changes in proteoglycans and glycosaminoglycans of patients with recurrent and nonrecurrent herniations of lumbar intervertebral discs. Material and Methods. Biochemical parameters of primary intervertebral disc hernias were prospectively investigated in 50 patients who underwent repeated surgery for recurrent hernia at the same level, and in 50 patients without recurrence. The quantity and quality of glycosaminoglycans in tissues of the hernia and surrounding annulus fibrosus were determined by analytical biochemistry methods. The properties of proteoglycans in the hernia tissue were studied by composite gel electrophoresis. Results. Type 1 recurrent hernia tissues are characterized by high content of water and glycosaminoglycans, but relatively few glycosaminoglycans tightly bound within the extracellular matrix (ECM) are retained in the tissue after its treatment with 4 M guanidinium chloride. Type 2 hernia tissues showed reduced content of water and glycosaminoglycans, and increased content of neutral hexoses. More than a half of them were tightly bound to ECM structures. On MRI evidence, Type 1 hernias correspond to Grade III disc degeneration, and Type 2 – to Grade IV. Biochemical profile of glycosaminoglycans in intervertebral disc tissues of the recurrence group reliably differed from that of the nonrecurrence group by the higher content of chondroitin sulfates and proteoglycans loosely bound to ECM structures. Conclusion. The tissue structure of nucleus pulposus and annulus fibrosus in recurrent and nonrecurrent intervertebral disc differs in qualitative and quantitative characteristics of glycosaminoglycans.