Predominant Glycosaminoglycan Research Articles

Characterization and Structural Analysis of a Novel Carbohydrate-Binding Module from Family 96 with Chondroitin Sulfate-Specific Binding Capacity.

Chondroitin sulfate (CS) is the predominant glycosaminoglycan within the human body and is widely applied in various industries. Carbohydrate-binding modules (CBMs) possessing the capacity for carbohydrate recognition are verified to be important tools for polysaccharide investigation. Only one CS-specific CBM, PhCBM100, has hitherto been characterized. In the present study, two CBM96 domains present in the same putative PL8_3 chondroitin AC lyase were discovered and recombinantly expressed. The results of microtiter plate assays and affinity gel electrophoresis assays showed that the two corresponding proteins, DmCBM96-1 and DmCBM96-2, bind specifically to CSs. The crystal structure of DmCBM96-1 was determined at a 2.20 Å resolution. It adopts a β-sandwich fold comprising two antiparallel β-sheets, showing structural similarities to TM6-N4, which is the founding member of the CBM96 family. Site mutagenesis analysis revealed that the residues of Arg27, Lys45, Tyr51, Arg53, and Arg157 are critical for CS binding. The characterization of the two CBM96 proteins demonstrates the diverse ligand specificity of the CBM96 family and provides promising tools for CS investigation.

A Concise Review of Extraction and Characterization of Chondroitin Sulphate from Fish and Fish Wastes for Pharmacological Application.

Chondroitin sulphate (CS) is one of the most predominant glycosaminoglycans (GAGs) available in the extracellular matrix of tissues. It has many health benefits, including relief from osteoarthritis, antiviral properties, tissue engineering applications, and use in skin care, which have increased its commercial demand in recent years. The quest for CS sources exponentially increased due to several shortcomings of porcine, bovine, and other animal sources. Fish and fish wastes (i.e., fins, scales, skeleton, bone, and cartilage) are suitable sources of CS as they are low cost, easy to handle, and readily available. However, the lack of a standard isolation and characterization technique makes CS production challenging, particularly concerning the yield of pure GAGs. Many studies imply that enzyme-based extraction is more effective than chemical extraction. Critical evaluation of the existing extraction, isolation, and characterization techniques is crucial for establishing an optimized protocol of CS production from fish sources. The current techniques depend on tissue hydrolysis, protein removal, and purification. Therefore, this study critically evaluated and discussed the extraction, isolation, and characterization methods of CS from fish or fish wastes. Biosynthesis and pharmacological applications of CS were also critically reviewed and discussed. Our assessment suggests that CS could be a potential drug candidate; however, clinical studies should be conducted to warrant its effectiveness.

Procedures to Evaluate the Role of Heparan Sulfate on the Reactivity of Resistance and Conductance Arteries Ex Vivo.

Evidence is emerging that disruption of the endothelial glycocalyx might contribute importantly to arterial dysfunction in the context of diabetes. One approach to assess the integrity of the endothelium and the vascular smooth muscle cell layer, in the absence of neural, humoral, and mechanical influences, is by measuring arterial vasomotion ex vivo. Here we describe a procedure to assess non-receptor-mediated vasoconstriction, receptor-mediated vasoconstriction, and endothelium-dependent and -independent vasodilation, in resistance and conductance arteries pressurized to 60mmHg. In addition to evaluating vasoreactivity using isobaric approaches, the same experimental set-up can be used to initiate a pressure gradient across the artery such that intraluminal, flow-mediated vasodilation can be measured. After recording endothelium-dependent vasodilation using isobaric or flow-mediated approaches, identical interventions can be completed in the presence of enzymes that cleave biologically active heparan sulfates into inactive disaccharide and oligosaccharide fragments to assess the contribution from: (a) endothelial-derived substances (e.g., nitric oxide via nitric oxide synthase inhibition); or (b) important components of the glycocalyx (e.g., removal of heparan sulfate via heparitinase III treatment). Here, we show that acute disruption of a predominant glycosaminoglycan i.e., heparan sulfate impairs intraluminal flow-mediated vasodilation in murine resistance arteries.

Enhanced Cellular Uptake and Sustained Transdermal Delivery of Collagen for Skin Regeneration

The present study reports a method for transporting high molecular weight collagen for skin regeneration. An independent engineered enzymatic vehicle that has the ability for efficient transdermal delivery of regenerative biomaterial was developed for tissue regeneration. Collagen has been well recognized as a skin regeneration molecule due to its interaction with the extracellular matrix to stimulate skin cell growth, proliferation, and differentiation. However, the transdermal delivery of collagen poses a significant challenge due to its high molecular weight as well as a lack of efficient approaches. Here, to improve the transdermal delivery efficiency, α-1,4-glycosidic hydrolase was engineered with genetically encoded 3,4-dihydroxy-L-phenylalanine, which enhanced its biological activity as revealed by microscale thermophoresis. The remodeled catalytic pocket resulted in enhanced substrate binding activity of the enzyme with a predominant glycosaminoglycan (chondroitin sulfate) present in the extracellular matrix of the skin. The engineered enzyme rapidly opened up the skin extracellular matrix fiber (15 min) to ferry collagen across the wall, without disturbing the cellular bundle architecture. Confocal microscopy indicated that macromolecules had diffused three times deeper into the engineered enzyme-treated skin than the native enzyme-treated skin. Gene expression, histopathology, and hematology analysis also supported the penetration of macromolecules. Cytotoxicity (mammalian cell culture) and in vivo (Caenorhabditis elegans and Rattus noryegicus) studies revealed that the congener enzyme could potentially be used as a penetration enhancer, which is of paramount importance for the multimillion cosmetic industries. Hence, it offers promise as a pharmaceutical enzyme for transdermal delivery bioenhancement and dermatological applications.

Hyaluronan Depolymerization by Megakaryocyte Hyaluronidase-2 Is Required for Thrombopoiesis

Hyaluronan is the predominant glycosaminoglycan component of the extracellular matrix with an emerging role in hematopoiesis. Modulation of hyaluronan polymer size is responsible for its control over cellular functions, and the balance of hyaluronan synthesis and degradation determines its molecular size. Although two active somatic hyaluronidases are expressed in mammals, only deficiency in hyaluronidase-2 (Hyal-2) results in thrombocytopenia of unknown mechanism. Our results reveal that Hyal-2 knockout mice accumulate hyaluronan within their bone marrow and within megakaryocytes, the cells responsible for platelet generation. Proplatelet formation by Hyal-2 knockout megakaryocytes was disrupted because of abnormal formation of the demarcation membrane system, which was dilated and poorly developed. Importantly, peptide-mediated delivery of exogenous hyaluronidase rescued deficient proplatelet formation in murine and human megakaryocytes lacking Hyal-2. Together, our data uncover a previously unsuspected mechanism of how hyaluronan and Hyal-2 control platelet generation.

Extraction, purification and characterisation of dermatan sulphate from bovine collagen waste liquor

The aim of this study was to extract, purify and characterise glycosaminoglycans present in bovine collagen waste liquor. Processing of bovine hides supplies collagen and gelatine to food, cosmetic and pharmaceutical industries generating abundant and unutilised waste streams that contain bioactive polysaccharides known as glycosaminoglycans. Some of these molecules, like heparin and dermatan sulphate, have known anti-thrombotic and anti-coagulant activities and are prescribed therapeutics. The predominant glycosaminoglycan in bovine collagen waste liquor, dermatan sulphate, was extracted and purified to greater than 93% w/w using only serial filtration and anion exchange chromatography. Bioactivity was measured using an in vitro assay revealing anti-thrombotic activity equivalent to commercial dermatan sulphate sourced from porcine mucosa. Fluorophore-assisted carbohydrate electrophoresis revealed a composition rich in the predominant mammalian disaccharide, uronic acid→N-acetyl-D-galactosamine-4-O-sulphate. Depolymerization and fractionation enriched for low molecular weight fragments with significantly improved anti-thrombotic activity. This study identifies an alternative source of anti-thrombotic dermatan sulphate that can be easily extracted without proteolysis or solvent precipitation that can be further processed into low molecular weight fractions with improved anti-thrombotic activity.

Hyaluronan - a functional and structural sweet spot in the tissue microenvironment.

Transition from homeostatic to reactive matrix remodeling is a fundamental adaptive tissue response to injury, inflammatory disease, fibrosis, and cancer. Alterations in architecture, physical properties, and matrix composition result in changes in biomechanical and biochemical cellular signaling. The dynamics of pericellular and extracellular matrices, including matrix protein, proteoglycan, and glycosaminoglycan modification are continually emerging as essential regulatory mechanisms underlying cellular and tissue function. Nevertheless, the impact of matrix organization on inflammation and immunity in particular and the consequent effects on tissue healing and disease outcome are arguably under-studied aspects of adaptive stress responses. Herein, we review how the predominant glycosaminoglycan hyaluronan (HA) contributes to the structure and function of the tissue microenvironment. Specifically, we examine the evidence of HA degradation and the generation of biologically active smaller HA fragments in pathological settings in vivo. We discuss how HA fragments versus nascent HA via alternate receptor-mediated signaling influence inflammatory cell recruitment and differentiation, resident cell activation, as well as tumor growth, survival, and metastasis. Finally, we discuss how HA fragmentation impacts restoration of normal tissue function and pathological outcomes in disease.

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.

The expression of glycosaminoglycans and proteoglycans in the uterine cervix of albino rats after local hyaluronidase infusion

Objective: To assess the local effect of hyaluronidase injection on the expression of glycosaminoglycans (GAGs) and proteoglycans (PGs) in the extracellular matrix of the uterine cervix from pregnant albino rats.Methods: Ten pregnant rats were divided into two groups on day 18 of pregnancy. The experimental group (Gexp) of rats received an intracervical infusion of 0.02 mL of hyaluronidase diluted to 1 mL with distilled water, whereas the control group (Gc) received 1 mL of distilled water. On day 20 of pregnancy, the pregnant rats were sacrificed and the uterine cervixes from all rats were then dissected. The qualitative expression of hyaluronic acid (HA) was assessed by immunohistochemistry and quantified by sandwich ELISA. To compare the quantitative GAG values between groups, a Student’s t-test for independent samples was performed. PGs were also assessed by immunohistochemical analysis.Results: The electrophoretic profile of newly synthesized radioactively labeled GAGs degraded by specific enzymes showed that there were two predominant GAGs in both Gc and Gexp, i.e. heparan sulfate (HS) and a mixture of hondroitin sulfate (CS) and dermatan sulfate (DS). The concentrations of GAGs showed a significant reduction of CS/DS (p < 0.004) and HS (p < 0.005) relative to Gc. HA staining was less intense in the lamina propria and area surrounding the blood vessels in Gexp compared to Gc. The HA contents were also significantly reduced (p < 0.012).Conclusions: Intracervical hyaluronidase infusion promoted a significant reduction in the concentration of sulfated GAGs as assessed by both qualitative (histochemical) and quantitative (fluorometric) measurements of HA.

The Two Phases of Heparin-Induced Thrombocytopenia (HIT): Early Monocyte/Tissue Factor (TF) Phase and Late Platelet Phase

Abstract 270HIT is an immune thrombocytopenic disorder associated with a high risk of thrombosis. Mechanistic studies have focused on circulating platelet factor 4 (PF4)/heparin complexes and on the subsequent activation of platelets. We and others have shown that binding of PF4 to cell surface glycosaminoglycans (GAGs) not only makes platelets critical targets in the pathogenesis of HIT, but monocytes and neutrophils as well. Our previous observations that monocyte depletion using clodronate-laden liposomes prior to induction of HIT in a passive immunization model mitigated the prothrombotic state, but paradoxically exacerbated initial thrombocytopenia, suggest that our understanding of the relationship between monocyte activation, platelet activation, thrombocytopenia and thrombosis is unsettled. Chondroitin sulfate is the predominant GAG expressed by platelets; therefore, they bind PF4 less avidly than monocytes, which have a cell surface rich in heparan and dermatan sulfates. Based on this cell-type difference in surface affinity for PF4, we hypothesized that: 1) monocytes and perhaps other vascular cells bind PF4 and form surface PF4/HIT antigenic complexes preferentially when compared with platelets, and 2) depletion of this high-affinity (monocyte) “sink” shifts PF4 binding to platelets making them more targeted. Flow cytometric analysis of whole mouse and human blood support these hypotheses as monocytes bind ∼100-fold more FITC-labeled human (h) PF4 than platelets or red blood cells and ∼10-fold more than neutrophils or lymphocytes. When isolated platelets and white blood cells are admixed, the amount of exogenously added hPF4 bound to platelets is inversely related to the leukocyte:platelet ratio. In vitro, exposure of whole blood to the HIT-like monoclonal antibody KKO plus recombinant hPF4 generated an intense platelet activation characterized by binding of annexin V and factor Xa, consistent with formation of coated platelets. Importantly, formation of coated platelets was attenuated by monocyte depletion from whole blood samples or by inhibition of thrombin by PPACK. We then infused KKO into transgenic mice expressing hPF4 and hFcgRIIA to induce HIT and followed the temporal profile of antibody binding to various cell types. Binding of KKO to monocytes was detected within 30 min of injection. These monocytes remained the predominant target over the first 4 hrs, after which binding decreased as the circulating monocytes were depleted. The mechanism and implications of this relatively late monocytopenia during HIT is under study. However, these data provide an explanation of how clodronate-laden monocyte depletion prior to inducing HIT exacerbated thrombocytopenia in the murine model of HIT, while decreasing the prothrombotic state: Early in the disease when PF4 is limited, monocytes selectively bind the PF4 and are targeted by HIT antibodies, which induces tissue factor and generates thrombin, but limits initial thrombocytopenia. Later, after induction of large amounts of TF, activated monocytes are cleared, shifting the target of antigen-antibody interactions to the surface of platelets, enhancing their response to available thrombin, thereby establishing a feed-forward prothrombotic cycle and more platelet clearance. In conclusion, we propose that HIT evolves from a monocyte-focused to a platelet-focused disease and that early intervention to prevent monocyte activation provides a new important potential therapeutic target for intervention at the earliest stages of disease recognition that may become less effective as time passes. Disclosures:Cines:Amgen Inc.: Consultancy; GlaxoSmithKline: Consultancy; Eisai: Consultancy.

828 URINE AND TISSUE GLYCOSAMINOGLYCANS AS BIOMARKERS FOR PAINFUL BLADDER SYNDROME/INTERSTITIAL CYSTITIS – INSIGHT ON PATHOPHYSIOLOGY?

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Interstitial Cystitis1 Apr 2012828 URINE AND TISSUE GLYCOSAMINOGLYCANS AS BIOMARKERS FOR PAINFUL BLADDER SYNDROME/INTERSTITIAL CYSTITIS – INSIGHT ON PATHOPHYSIOLOGY? Marcos Lucon, Roberto Soler, Joao R.M. Martins, Elsa K. Yoko, Helena B. Nader, Miguel Srougi, and Bruschini Homero Marcos LuconMarcos Lucon Sao Paulo, Brazil More articles by this author , Roberto SolerRoberto Soler Sao Paulo, Brazil More articles by this author , Joao R.M. MartinsJoao R.M. Martins Sao Paulo, Brazil More articles by this author , Elsa K. YokoElsa K. Yoko Sao Paulo, Brazil More articles by this author , Helena B. NaderHelena B. Nader Sao Paulo, Brazil More articles by this author , Miguel SrougiMiguel Srougi Sao Paulo, Brazil More articles by this author , and Bruschini HomeroBruschini Homero Sao Paulo, Brazil More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.918AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urothelial glycosaminoglycan (GAG) layer may play a role in the etiology of painful bladder syndrome/interstitial cystitis (IC). GAG in urine and bladder tissue of those patients was investigated. METHODS Urine was collected from 11 female patients with a clinical diagnosis of IC by bladder diary and a self-reported validated questionnaire, and cold-cup bladder biopsies were obtained before hydrodistension. Urine and bladder biopsies were also obtained from 11 female patients with pure SUI, as controls. Sulfated GAG (S-GAG) and hyaluronic acid (HA) were measured in the urine and tissue samples were used for proteoglycans, HA and extra-cellular matrix proteins immunostainings; and analyses of S-GAG levels and gene expression of HA synthases and hyaluronidase. RESULTS Urine analysis of S-GAG in IC patients and controls exhibited a different pattern when compared to tissue evaluation. Urine S-GAG concentration was lower in IC patients (0.45±0.11 x 0.62±0.13 μg/mg creatinine, p=0.01); while a similar concentration was found in the bladder tissue: 3.3 (0.58-7.08) x 2.7 (0.15-5.3) μg/mg (p=0.62). Interestingly, their pattern of distribution remained the same, being chondroitin sulfate the predominant GAG. Decorin, a chondroitin and dermatan sulfate proteoglycan, was more intensely expressed in the urothelium of IC patients, while the expression of syndecan-4, a heparan sulfate proteoglycan, was scarcer in the urothelium of IC patients. Fibronectin was highly expressed in IC patients in the layers underneath the urothelium. These findings may represent local inflammation, superficial urothelial desquamation and extracellular matrix remodeling. Urine HA levels was similar between the groups (1.71±2.06 x 2.46±2.07 ng/mg creatinine; p=0.48). However, HA labeling in the urothelial and suburothelial layers was more intense without a corresponding increasing in the expression of its receptor CD 44. This may represent a shift in HA turnover with consequent deposition of HA in the interstitium, where interacting with other matrix components, it could participate in remodeling and recovery of the urothelium. HA synthases 1, 2 and 3 and hyaluronidase gene expression was consistently lower in PBS/IC patients, which may also suggest changes in HA turnover. CONCLUSIONS Analyses of GAG in patients with IC may be important in understanding the pathophysiology of the disease. Since changes in HA synthases and hyaluronidase gene expression were consistent among PBS/IC patients, they could possibly be a marker of the disease. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e338 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Marcos Lucon Sao Paulo, Brazil More articles by this author Roberto Soler Sao Paulo, Brazil More articles by this author Joao R.M. Martins Sao Paulo, Brazil More articles by this author Elsa K. Yoko Sao Paulo, Brazil More articles by this author Helena B. Nader Sao Paulo, Brazil More articles by this author Miguel Srougi Sao Paulo, Brazil More articles by this author Bruschini Homero Sao Paulo, Brazil More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Glycosaminoglycans in the accessory sex glands, testes and seminal plasma of alpaca and ram

The viscous nature of alpaca semen limits its use in cryopreservation and other assisted reproductive technologies. The cause and source of this viscosity is unknown although it has been postulated, but never proven, that glycosaminoglycans (GAGs) secreted by the bulbourethral gland are responsible. The present study investigated the concentration and composition of GAGs in alpaca seminal plasma, testes, bulbourethral gland and prostate gland and compared them to those in the ram to determine the relationship between seminal plasma GAGs and viscosity and to identify the source of seminal plasma GAGs. Alpaca seminal plasma contained more GAGs than ram (P<0.001) and the predominant GAG, keratan sulfate, was correlated with viscosity (P=0.05, R(2)=0.2635). The alpaca bulbourethral gland contained most GAGs compared with prostate or testis (P<0.001). In the ram, the prostate contained most GAGs. These findings suggest that GAGs, particularly keratan sulfate, may be the cause of seminal plasma viscosity in alpacas, and that the seminal plasma GAGs originate from the bulbourethral gland.

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.

Decorin and biglycan expression: its relation with endothelial heterogeneity.

Decorin and biglycan proteoglycans play important roles in the organization of the extracellular matrix, and in the regulation of cell adhesion and migration. Given morphological and functional endothelial heterogeneity, information is needed regarding whether endothelial cells (ECs) from different vascular beds possess different profiles of proteoglycan constituents of the basement membranes. Here, we report that endothelia from different murine organs and EC lines derived thereof produce and secrete different patterns of proteoglycans. A faint colocalization between decorin and PECAM/CD31 was found on tissue sections from mouse heart, lung and kidney by immunofluorescence. Three EC lines derived from these organs produced decorin (100-kDa) and its core protein (45-kDa). Extracellular decorin recognition in culture supernatant was only possible after chondroitin lyase digestion suggesting that the core protein of secreted proteoglycan is more encrypted by glycosaminoglycans than the intracellular one. Heart and lung ECs were able to produce and release decorin. Kidney ECs synthesized the proteoglycan and its core protein but no secretion was detected in culture supernatants. Although biglycan production was recorded in all EC lines, secretion was almost undetectable, consistent with immunofluorescence results. In addition, no biglycan secretion was detected after EC growth supplement treatment, indicating that biglycan is synthesized, secreted and quickly degraded extracellularly by metalloproteinase-2. Low molecular-mass dermatan sulfate was the predominant glycosaminoglycan identified bound to the core protein. ECs from different vascular beds, with differences in morphology, physiology and cell biology show differences in the proteoglycan profile, extending their heterogeneity to potential differences in cell migration capacities.

Uterine prolapse: evaluation of glycosaminoglycans in postmenopausal women after estrogen therapy

Objective To evaluate glycosaminoglycans (GAGs) in the parametrium, paraurethral tissue and vaginal apex in postmenopausal women with uterine prolapse and to evaluate the effect of 30-day estrogen therapy in these patients.Material and methods Double-blind trial of estrogen and placebo in 40 women with a control group of 20 premenopausal women without uterine prolapse. Twenty postmenopausal women with prolapse formed a second group and were treated with placebo for 30 days before vaginal hysterectomy. The third group included 20 postmenopausal women with prolapse who received 0.625 mg oral conjugated estrogens for 30 days before vaginal hysterectomy. Samples of the parametrium, vaginal apex and paraurethral tissue were obtained during surgery.Results Hyaluronic acid was the predominant GAG detected, followed by dermatan sulfate, chondroitin sulfate and heparan sulfate. In postmenopausal women with prolapse, we did not observe significant differences in total GAGs compared to the control group. However, hyaluronic acid was increased in the parametrium of women receiving estrogen compared to those treated with the placebo (2033.39 ± 3037.90 mg/g vs. 587.87 ± 697.89 mg/g, respectively; p == 0.041).Conclusions There are differences in GAGs in the parametrium, paraurethral tissue and vaginal apex between women in premenopause and those in the postmenopause period. Therefore, 30-day estrogen therapy produces significant differences in levels of hyaluronic acid, dermatan sulfate and chondroitin sulfate.

Role of keratan sulphate (sulphated poly -N-acetyllactosamine repeats) in keratoconic cornea, histochemical, and ultrastructural analysis

Keratan sulphate (KS) is the predominant glycosaminoglycan (GAG) present in the corneal stroma where it is thought to regulate collagen fibril diameter. In this study we investigated the distribution of KS in normal and keratoconic corneas. Four normal, one mild, and four severe keratoconic corneas were used for the study. Distribution of keratan sulphate proteoglycans (KS-PG) was investigated using a primary monoclonal antibody (5-D-4) that recognizes disulphated disaccharides in the poly-N-acetyllactosamine repeats of KS. The immuno-reactivity of 5-D-4 was analyzed by immunohistochemistry and immuno-electron microscopy. Immuno-histochemistry showed diffuse 5-D-4 staining in keratoconic cornea compared to the punctuate staining in normal corneas. In the single cornea with mild keratoconus, immunogold microscopy revealed a very high density of KS-PG staining, especially in the posterior stroma, compared to severe keratoconic and normal cornea. The amount of KS-PG in the stroma in severe keratoconus was slightly less compared to the normal cornea. In the mild keratoconic cornea, a higher quantity of KS-PG was present around the keratocytes. In severe keratoconic corneas, a higher quantity of KS-PG was present within the keratocytes compared to normal cornea. The finding of an altered expression of KS in our keratoconic corneas, in particular the strong expression of KS in keratocytes, is in keeping with reports of an altered expression of proteoglycan metabolism in keratoconus. KS-PG plays an important role in stromal collagen fibril assembly and a dysregulation of KS-PG synthesis or catabolism could explain changes in collagen fibril spacing and diameter, which we have reported elsewhere.

Analysis of Glycosaminoglycans in the Parametrium and Vaginal Apex of Women with and without Uterine Prolapse

Pelvic organ prolapse (POP) is a downward descent of pelvic organs that results in protrusions of the vagina, the uterus, or both. The cause of this disorder is likely to be multifactorial, attributable to a combination of risk factors, especially connective tissue disorders. Our objective was to characterize and quantify a component of the extracellular matrix (ECM)-sulfated glycosaminoglycan (GAG)-in the parametrium and vaginal apex of women with and without uterine prolapse. Parametrium and vaginal apex tissue was obtained from 42 women who underwent surgery. Patients underwent a physical examination and were divided into groups according to the type of genital prolapse. Standard biopsies were taken during surgery and were assessed by biochemical methods. GAGs were obtained by proteolysis. The relative concentration of GAGs was determined by densitometry. Data were compared using an independent sample t-test or chi(2) test. In both groups (with and without prolapse) and in both types of tissue, dermatan sulfate (DS) was the most predominant glycosaminoglycan, followed by chondroitin sulfate (CS) and heparan sulfate (HS). We did not observe significant differences in the total amounts of GAGs, DS, CS, or HS. This study did not show altered biochemical characteristics in the ECM of parametrium and vaginal apex tissue of women either with or without uterine prolapse.

Glycosaminoglycans and glycoconjugates in the adult anuran integument (Lithobates catesbeianus)

Glycosaminoglycans (GAGs) from the integument of Lithobates catesbeianus were biochemically characterized and histochemically localized. Moreover, carbohydrate distribution was investigated using conventional and lectin histochemistry at light microscopy. Hyaluronan (HA), dermatan sulfate (DS) and a heparanoid were found in the integument. Sulfated and carboxylated GAGs were visualized in the Eberth-Katschenko (EK) layer, in the mucous glands, in the hypodermis as well as in the mast cells. Furthermore, glucose and galactose were identified in the integument through thin layer chromatography (TLC) assays. N-Acetyl-β-glucosamine residues were identified in the mucous glandular cells, between the corneum and spinosum strata, in the subepidermal region, and in the EK layer. N-Acetyl-galactosamine residues were evident in the EK layer, corresponding to a residue of the dermatan sulfate chain, which may be related to the collagenous fiber arrangement. These glycoconjugates occurred as secretory glandular products and as dermal structural elements. Moreover, HA and DS are the predominant GAGs in the L. catesbeianus integument. Considering the importance of glycoconjugates, they play a significant role to the integrity of the skin, providing mechanical support for integument cells. In addition, they are important to the water regulation mechanisms, since L. catesbeianus is preferably aquatic.

Structural and biochemical aspects of keratan sulphate in the cornea

Keratan sulphate (KS) is the predominant glycosaminoglycan (GAG) in the cornea of the eye, where it exists in proteoglycan (PG) form. KS-PGs have long been thought to play a pivotal role in the establishment and maintenance of the array of regularly-spaced and uniformly- thin collagen fibrils which make up the corneal stroma. This characteristic arrangement of fibrils allows light to pass through the cornea. Indeed, perturbations to the synthesis of KS-PG core proteins in genetically altered mice lead to structural matrix alterations and corneal opacification. Similarly, mutations in enzymes responsible for the sulphation of KS-GAG chains are causative for the inherited human disease, macular corneal dystrophy, which is manifested clinically by progressive corneal cloudiness starting in young adulthood.

The mRNA expression of prostaglandin E receptors EP 2 and EP 4 and the changes in glycosaminoglycans in the sheep cervix during the estrous cycle

Transcervical artificial insemination in sheep is limited by the inability to completely penetrate the cervix with an inseminating pipette. Penetration is partially enhanced at estrus due to a degree of cervical relaxation, which is probably regulated by cervical prostaglandin synthesis and extracellular matrix remodeling. Prostaglandin E 2 acts via prostaglandin E receptors EP 1 to EP 4, and EP 2 and EP 4 stimulate smooth muscle relaxation and glycosaminoglycan synthesis. This study investigated the expression of EP 2 and EP 4 mRNA and glycosaminoglycans in the sheep cervix during the estrous cycle. Sheep cervices were collected prior to, during, and after the luteinizing hormone (LH) surge and during the luteal phase. The mRNA expression of EP 2 and EP 4 was determined by in situ hybridization, glycosaminoglycan composition was assessed by Alcian blue staining, and hyaluronan concentration was investigated by ELISA. The expression of EP 2 mRNA was greatest prior to the LH surge ( P = 0.02), although EP 2 and EP 4 were expressed throughout the estrous cycle. Hyaluronan was the predominant glycosaminoglycan, and hyaluronan content increased prior to the LH surge ( P < 0.05). Cervical EP 2 mRNA expression changed throughout the estrous cycle and was greatest prior to the LH surge. We propose that prostaglandin E 2 binds to EP 2 and EP 4 stimulating hyaluronan synthesis, which may cause remodeling of the cervical extracellular matrix, culminating in cervical relaxation.